fura-2 Search Results


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  • 99
    Thermo Fisher fura 2 am
    Basal- and glucose-stimulated insulin release and intracellular Ca 2+ levels in PHPT-1 +/+ and PHPT-1 −/− perifused islets. PHPT-1 +/+ and PHPT-1 −/− islets were cultured overnight in 10 mmol/L ( A i and ii) or 2 mmol/L ( B i–iv) glucose. A i and B i: Basal, glucose (10 mmol/L), and amino acid mixture (AAM) (4.0 mmol/L) induced Ca 2+ flux in PHPT-1 +/+ and PHPT-1 −/− islets determined by assessing 340-to-380 nm <t>Fura-2</t> <t>acetoxymethylester</t> emission ratios after subtracting background as indicated. The K ATP channel inhibitor glyburide (Glyb) and K ATP channel activator diazoxide (Diaz) were added at the end of experiments in A i to demonstrate that Ca 2+ flux was dependent on K ATP channel activity. B ii: KCl (30 mmol/L) was added at the end of the experiment to induce maximal Ca 2+ flux by depolarization of the membrane potential. A ii and B ii–iv: Basal, AAM, and glucose-induced insulin release was assessed in PHPT-1 +/+ and PHPT-1 −/− islets after perfusion with AAM (0–12 mmol/L) or glucose (G; 0–15, 25 mmol/L) as indicated. B iii and B iv: Magnification of the results shown in B ii. KCl (30 mmol/L) was added at the end of the experiment. Results are presented as ±SE per 150 islets. a.u., arbitrary units.
    Fura 2 Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fura 2 am
    Impact of mutations on the hydrogen peroxide-mediated TRPM2 activation. HEK293 cells were transiently transfected with an expression vector for EGFP and either human wild-type TRPM2 or a channel carrying the indicated mutation. At 24 h post transfection, cells were loaded with <t>Fura2</t> and subjected to calcium imaging. At the indicated time, either buffer or hydrogen peroxide (final concentration 1 mM) was added. Analysis was restricted to EGFP-positive cells. For differentiation between responding and non-responding cells, the indicated threshold of 0.7 was chosen.
    Fura 2 Am, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Dojindo Labs fura 2 am
    Effects of nicardipine-containing media on STOCs and caffeine-induced responses. (a) Effects of nicardipine-containing external solutions on STOCs and I CAF at a V H of −20 mV. (b) Effects of nicardipine-containing external solutions on <t>fura-2</t> microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while striped bars indicate application of nicardipine. Dotted lines indicate the zero current level.
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    99
    Thermo Fisher fura 2 calcium imaging calibration kit
    Cellular stress from AS aggregates causes early reduction in cytosolic calcium followed by later increase Cytosolic Ca 2+ levels were quantified by the Ca 2+ sensor <t>Fura‐2</t> and converted to absolute concentrations using the Fura‐2 Calcium Imaging Calibration Kit. The AS aggregation inhibitor ASI‐1D (20 μM) was used to validate that phenotypes were aggregate‐dependent. Statistical analyses were performed using one‐way ANOVA multiple comparisons with Sidak post hoc test. Mitotic OLN‐t40‐AS cells were transfected with p25α and the fluorescent transfection marker tdTomato. OLN‐t40‐AS cells transfected with tdTomato and empty expression vectors served as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0001, ** P = 0.0002, # P = 0.0011, ## P = 0.0033). The average Ca 2+ level of individual experiments was calculated by measuring > 50 or more tdTomato expressing cells. Non‐mitotic SH‐SY5Y cells were generated by treatment with retinoic acid (RA; 10 μM) for 2 days, after which AS expression was induced by removal of doxycycline (dox) and cytosolic Ca 2+ measured after 5 days and 10 days of AS expression. See timeline under bars. Cells induced to express β‐galactosidase (b‐gal) upon dox removal were used as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 4 (* P = 0.0005, ** P = 0.0005, # P = 0.0062, ## P = 0.0055). The average Ca 2+ level was calculated by measuring > 200 randomly selected cells in each experiment. Primary hippocampal neurons were isolated from new‐born (P0) mice expressing human AS under the mThy1 promoter and wild‐type (wt) littermates. Cytosolic Ca 2+ was measured after 5 days in vitro (5 DIV) and 14 days in vitro culture (14 DIV). See timeline under graphs. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.002, ** P = 0.0007, # P = 0.0071, ## P = 0.0455). The average cellular Ca 2+ is based on > 500 neurons per experiment. Cytosolic Ca 2+ in SHSY5Y cells as in (B) measured every second day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0377, ** P = 0.0057, *** P = 0.03, # P = 0.045, ## P = 0.0229). Cytosolic Ca 2+ in primary hippocampal neurons as in (C) measured every third day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.01, ** P = 0.0073, # P = 0.0058).
    Fura 2 Calcium Imaging Calibration Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fura 2 pentapotassium salt
    Cellular stress from AS aggregates causes early reduction in cytosolic calcium followed by later increase Cytosolic Ca 2+ levels were quantified by the Ca 2+ sensor <t>Fura‐2</t> and converted to absolute concentrations using the Fura‐2 Calcium Imaging Calibration Kit. The AS aggregation inhibitor ASI‐1D (20 μM) was used to validate that phenotypes were aggregate‐dependent. Statistical analyses were performed using one‐way ANOVA multiple comparisons with Sidak post hoc test. Mitotic OLN‐t40‐AS cells were transfected with p25α and the fluorescent transfection marker tdTomato. OLN‐t40‐AS cells transfected with tdTomato and empty expression vectors served as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0001, ** P = 0.0002, # P = 0.0011, ## P = 0.0033). The average Ca 2+ level of individual experiments was calculated by measuring > 50 or more tdTomato expressing cells. Non‐mitotic SH‐SY5Y cells were generated by treatment with retinoic acid (RA; 10 μM) for 2 days, after which AS expression was induced by removal of doxycycline (dox) and cytosolic Ca 2+ measured after 5 days and 10 days of AS expression. See timeline under bars. Cells induced to express β‐galactosidase (b‐gal) upon dox removal were used as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 4 (* P = 0.0005, ** P = 0.0005, # P = 0.0062, ## P = 0.0055). The average Ca 2+ level was calculated by measuring > 200 randomly selected cells in each experiment. Primary hippocampal neurons were isolated from new‐born (P0) mice expressing human AS under the mThy1 promoter and wild‐type (wt) littermates. Cytosolic Ca 2+ was measured after 5 days in vitro (5 DIV) and 14 days in vitro culture (14 DIV). See timeline under graphs. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.002, ** P = 0.0007, # P = 0.0071, ## P = 0.0455). The average cellular Ca 2+ is based on > 500 neurons per experiment. Cytosolic Ca 2+ in SHSY5Y cells as in (B) measured every second day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0377, ** P = 0.0057, *** P = 0.03, # P = 0.045, ## P = 0.0229). Cytosolic Ca 2+ in primary hippocampal neurons as in (C) measured every third day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.01, ** P = 0.0073, # P = 0.0058).
    Fura 2 Pentapotassium Salt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Olympus fura 2
    Emission spectra of RES and <t>Fura-2.</t> Separate measurements (A) of 340 nm (Ca 2+ -bound, blue) and 380 nm (Ca 2+ -free, orange) signal intensity in MDA cells with 5 µM TG and 100 µM RES treatments at 1 min and 6 min, respectively, as indicated by the arrows. This data (A) is representative of all experiments conducted at these concentrations. Emission spectra (B) in 1 mM Ca 2+ solution when excited at 340 nm of 100 µM RES (blue), and 5 µM Fura-2 (yellow).
    Fura 2, supplied by Olympus, used in various techniques. Bioz Stars score: 93/100, based on 391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher mag fura 2
    Overexpressed wtTG2 appears in the mitochondria and induces enhanced mitochondrial Ca 2+ accumulation. (A) Time dependent changes in the mitochondrial Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Rhod-2/AM. (B) Time dependent changes in the cytosolic Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by <t>Fura-2/AM.</t> (C) Time dependent changes in the mitochondrial and cytoplasmic TG2 expressions of Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Western blot analysis. Mitochondrial HSP60 and cytoplasmic β-actin were used as loading controls; while β-tubulin was used to check for cytoplasmic contamination of mitochondria. (D) Time dependent changes in the mitochondrial and cytoplasmic TG2 activities of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment. (E) Confocal images taken at 16 h following Dox treatment show Tet-on wtTG2 cells expressing TG2 (green) colocalized with the mitochondrial calcium indicator Rhod-2/AM (red). Scale bar = 5 µM. All the data presented represent mean±S.D. of at least three determinations. Significantly different from the TG2C277S cell line detected at the same time point (* P
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    93
    Sutter Instrument fura 2
    Brain slices were incubated in a stage top chamber with 5%CO2 at 37°C. <t>Fura-2</t> images were obtained for brain slices at 5s intervals for a total of 15min. Time lapse series of P4 GFP-expressing OPCs in the Dorsolateral subventricular zone ( SVZ ) ( A ) and in the Corpus Callosum ( CC ) ( B ). Each frame represents a single section of a Fura-2 time lapse experiment. An increased Fura-2 fluorescence ratio is indicated by warmer colors. Time is denoted in minutes in the upper right corner and the area of the CC and SVZ is indicated in the inset. LV : lateral ventricle. Scale bar ( A ) 100μm, ( B ) 50μm. ( C and D ) VOCC activity was examined in P4 GFP-expressing OPCs from the SVZ area and OLs from the CC. Note that each trace corresponds to a single cell and the time of addition of external solution containing high K + is indicated by the horizontal bars. ( E ) K + -induced Ca ++ uptake in P4 OPCs from the SVZ was abolished in 25μM verapamil, 25μM nifedipine and in the absence of external Ca ++ (-Ca ++ ). The graph show the average amplitude calculated from the responding cells, expressed as percentage of change of the emission intensities. ( F ) VOCC activity was examined in P4 and P8 GFP-expressing OPCs from the SVZ area and OLs from the CC. The graph show the average maximal peak values and plateau values during minutes 9-11, calculated from the responding cells, expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). *p
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    92
    Biotium fura 2
    Emission spectra of RES and <t>Fura-2.</t> Separate measurements (A) of 340 nm (Ca 2+ -bound, blue) and 380 nm (Ca 2+ -free, orange) signal intensity in MDA cells with 5 µM TG and 100 µM RES treatments at 1 min and 6 min, respectively, as indicated by the arrows. This data (A) is representative of all experiments conducted at these concentrations. Emission spectra (B) in 1 mM Ca 2+ solution when excited at 340 nm of 100 µM RES (blue), and 5 µM Fura-2 (yellow).
    Fura 2, supplied by Biotium, used in various techniques. Bioz Stars score: 92/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Dojindo Labs fura 2 acetoxymethyl ester
    J.SL1 cells lack expression of Shc and are defective in IL-2 production. ( A ) Cell lysates of Jurkat and J.SL1 cells were analyzed by Western blots with the antibodies shown on the side of each panel. ( B ) Northern blot for lck and shc mRNA. Total RNA from Jurkat and J.SL1 cells was analyzed by using the cDNA fragment for lck ( Left ) or shc ( Right ). ( C ) IL-2 production by Jurkat and J.SL1 cells. Cells were cultured 16 h with (TCR) or without (−) stimulation by anti-TCR ( Left ) or with PMA and ionomycin (P+I, Right ). The amount of IL-2 in the culture supernatant from each sample was determined by ELISA. A representative result of several experiments is shown. ( D ) Surface expression of CD69 and CD25 on Jurkat and J.SL1 cells. Jurkat and J.SL1 cells were treated as described for C , and expression of CD69 and CD25 were examined by using FITC-conjugated antibodies. The dark lines show fluorescence levels detected from activated cells, and gray lines show that from unstimulated cells. ( E ) Induction of intracellular Ca 2+ in Jurkat and J.SL1 cells. Jurkat ( Left ) and J.SL1 ( Right ) cells were loaded with <t>Fura-2</t> <t>acetoxymethyl</t> ester, and the cells were stimulated with anti-TCR at the times shown by the arrow above each panel. The level of Ca 2+ was determined on the basis of the ratio between fluorescence from excitation at 340 nm to that from excitation at 380 nm.
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    84
    Thermo Fisher 㠎 â 1 4 m fura 2
    J.SL1 cells lack expression of Shc and are defective in IL-2 production. ( A ) Cell lysates of Jurkat and J.SL1 cells were analyzed by Western blots with the antibodies shown on the side of each panel. ( B ) Northern blot for lck and shc mRNA. Total RNA from Jurkat and J.SL1 cells was analyzed by using the cDNA fragment for lck ( Left ) or shc ( Right ). ( C ) IL-2 production by Jurkat and J.SL1 cells. Cells were cultured 16 h with (TCR) or without (−) stimulation by anti-TCR ( Left ) or with PMA and ionomycin (P+I, Right ). The amount of IL-2 in the culture supernatant from each sample was determined by ELISA. A representative result of several experiments is shown. ( D ) Surface expression of CD69 and CD25 on Jurkat and J.SL1 cells. Jurkat and J.SL1 cells were treated as described for C , and expression of CD69 and CD25 were examined by using FITC-conjugated antibodies. The dark lines show fluorescence levels detected from activated cells, and gray lines show that from unstimulated cells. ( E ) Induction of intracellular Ca 2+ in Jurkat and J.SL1 cells. Jurkat ( Left ) and J.SL1 ( Right ) cells were loaded with <t>Fura-2</t> <t>acetoxymethyl</t> ester, and the cells were stimulated with anti-TCR at the times shown by the arrow above each panel. The level of Ca 2+ was determined on the basis of the ratio between fluorescence from excitation at 340 nm to that from excitation at 380 nm.
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    93
    Collaborative Drug Discovery Inc fura 2
    ATP (0.1 mM) produced a prompt increase in the <t>fura</t> 2 fluorescence ratio in MDCK monolayers perfused with solution containing 2 mM Ca 2+ ( top, left ). This was due in part to increased Ca 2+ influx because ATP stimulated rapid quenching of fura 2 by extracellular Mn 2+ (2 mM) in the absence of extracellular Ca 2+ ( top, right ). ATP treatment in Ca 2+ -free solution also increased the fluorescence ratio, suggesting mobilization of intracellular Ca 2+ stores ( bottom ). This was followed by a second fluorescence peak when extracellular calcium was restored, consistent with Ca 2+ influx. A single curve is shown that represents the mean ratio derived from the number of cells indicated on the graphs.
    Fura 2, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 93/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biotium fura 2 am
    SU-DHL-4 cells are protected from BIRD-2-triggered apoptosis by genetically manipulating the IP 3 signaling pathway. a Examples of flow cytometry analysis showing the percentage of GFP-positive transfected SU-DHL-4 cells, visible as a shift in the BL-1 (515–545 nm) channel, while the values in the BL-3 (665–715 nm) channel remained unaffected. b Representative flow cytometry analysis of BIRD-2-induced apoptosis in SU-DHL-4 cells transfected with the IP 3 sponge or a control vector compared to mock-transfected cells. Apoptosis was detected via Annexin V-APC-positive staining (RL1 + = red laser, see Method section) of the cells. c Quantification of the apoptotic fraction (%) after treatment with 10 µM BIRD-2 (red histogram in panel b ) or vehicle (black histogram panel b ) in mock-transfected SU-DHL-4 cells or cells transfected with the IP 3 sponge or a control vector. Apoptotic cells were identified as the Annexin V-APC-positive fraction (RL1 + ). Data are represented as mean ± SEM of 3 independent experiments. d Single-cell cytosolic Ca 2+ measurements performed in SU-DHL-4 cells utilizing <t>Fura-2</t> AM. Cells were transfected with an IP 3 sponge vector or with an empty vector as negative control condition (pcDNA3.1). The addition of 10 µM BIRD-2 is indicated by the arrow. Data are represented as the average ± SEM of 3 independent experiments ( n > 100 cells/condition)
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    90
    Thermo Fisher bis fura 2
    rgs3 impacts segmentation stage Ca 2+ dynamics. Zebrafish embryos injected with <t>Fura-2</t> were oriented in a dorsal posterior view. Representative ratio images, pseudocolored with low Ca 2+ represented by blue, and high Ca 2+ represented by yellow/red (A–C,F–N). During somitogenesis, Ca 2+ transients are identified as a local short-lived increase in intracellular Ca 2+ levels. A region of interest (ROI) is noted by a dashed circle highlighting a representative Ca 2+ transient (A–C). In the ROI from time 0s to time 15s, an increase in Ca 2+ levels is observed (B) that subsides by time 30s (C). The number of transients as a function of developmental age (D). Table depicting the average number of Ca 2+ transients per hour from 6 to 12 somite stage for each treatment (E). Representative ratio images of 5 somite stage (F), 7 somite (G) and 10 somite stage (H) wt embryos taken from Video S1 . Representative ratio images of 5 somite (I), 7 somite (J) and 10 somite stage (K) rgs3 MOsa injected embryo taken from Video S2 . Representative ratio images of 5 somite (L), 7 somite stage (M) and 10 somite stage (N) wnt5b MO injected embryo taken from Video S4 . Red arrowheads indicate large Ca 2+ transients in rgs3 morphant embryos (I–K) that are not observed in wt (F–H) or wnt5b morphant embryos (L–N).
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    Image Search Results


    Basal- and glucose-stimulated insulin release and intracellular Ca 2+ levels in PHPT-1 +/+ and PHPT-1 −/− perifused islets. PHPT-1 +/+ and PHPT-1 −/− islets were cultured overnight in 10 mmol/L ( A i and ii) or 2 mmol/L ( B i–iv) glucose. A i and B i: Basal, glucose (10 mmol/L), and amino acid mixture (AAM) (4.0 mmol/L) induced Ca 2+ flux in PHPT-1 +/+ and PHPT-1 −/− islets determined by assessing 340-to-380 nm Fura-2 acetoxymethylester emission ratios after subtracting background as indicated. The K ATP channel inhibitor glyburide (Glyb) and K ATP channel activator diazoxide (Diaz) were added at the end of experiments in A i to demonstrate that Ca 2+ flux was dependent on K ATP channel activity. B ii: KCl (30 mmol/L) was added at the end of the experiment to induce maximal Ca 2+ flux by depolarization of the membrane potential. A ii and B ii–iv: Basal, AAM, and glucose-induced insulin release was assessed in PHPT-1 +/+ and PHPT-1 −/− islets after perfusion with AAM (0–12 mmol/L) or glucose (G; 0–15, 25 mmol/L) as indicated. B iii and B iv: Magnification of the results shown in B ii. KCl (30 mmol/L) was added at the end of the experiment. Results are presented as ±SE per 150 islets. a.u., arbitrary units.

    Journal: Diabetes

    Article Title: Regulation of KATP Channel Trafficking in Pancreatic β-Cells by Protein Histidine Phosphorylation

    doi: 10.2337/db17-1433

    Figure Lengend Snippet: Basal- and glucose-stimulated insulin release and intracellular Ca 2+ levels in PHPT-1 +/+ and PHPT-1 −/− perifused islets. PHPT-1 +/+ and PHPT-1 −/− islets were cultured overnight in 10 mmol/L ( A i and ii) or 2 mmol/L ( B i–iv) glucose. A i and B i: Basal, glucose (10 mmol/L), and amino acid mixture (AAM) (4.0 mmol/L) induced Ca 2+ flux in PHPT-1 +/+ and PHPT-1 −/− islets determined by assessing 340-to-380 nm Fura-2 acetoxymethylester emission ratios after subtracting background as indicated. The K ATP channel inhibitor glyburide (Glyb) and K ATP channel activator diazoxide (Diaz) were added at the end of experiments in A i to demonstrate that Ca 2+ flux was dependent on K ATP channel activity. B ii: KCl (30 mmol/L) was added at the end of the experiment to induce maximal Ca 2+ flux by depolarization of the membrane potential. A ii and B ii–iv: Basal, AAM, and glucose-induced insulin release was assessed in PHPT-1 +/+ and PHPT-1 −/− islets after perfusion with AAM (0–12 mmol/L) or glucose (G; 0–15, 25 mmol/L) as indicated. B iii and B iv: Magnification of the results shown in B ii. KCl (30 mmol/L) was added at the end of the experiment. Results are presented as ±SE per 150 islets. a.u., arbitrary units.

    Article Snippet: Fura-2 acetoxymethylester (Life Technologies) was used as calcium indicator ( ).

    Techniques: Cell Culture, Activity Assay

    Physiological stretch alters Ca 2+ entry in heart VCMs are first attached to glass cardiac fingers (see Methods ). VCMs are loaded with Fura-2 AM (5 μ M, 15 min). A , extracellular Ca 2+ (1.8 mM) is replaced with equimolar Mn 2+ which enters the cell via sarcolemmal Ca 2+ channels and quenches Fura-2 florescence (see Slope 1). During a 2 Hz cyclic stretch, elevated Ca 2+ influx increases the quench rate (see Slope 2). B , analysis of Ca 2+ influx rates. Statistics: MDX compared to WT (* P

    Journal: The Journal of physiology

    Article Title: Quantitative tests reveal that microtubules tune the healthy heart but underlie arrhythmias in pathology

    doi: 10.1113/JP277083

    Figure Lengend Snippet: Physiological stretch alters Ca 2+ entry in heart VCMs are first attached to glass cardiac fingers (see Methods ). VCMs are loaded with Fura-2 AM (5 μ M, 15 min). A , extracellular Ca 2+ (1.8 mM) is replaced with equimolar Mn 2+ which enters the cell via sarcolemmal Ca 2+ channels and quenches Fura-2 florescence (see Slope 1). During a 2 Hz cyclic stretch, elevated Ca 2+ influx increases the quench rate (see Slope 2). B , analysis of Ca 2+ influx rates. Statistics: MDX compared to WT (* P

    Article Snippet: Here myocytes were loaded with 5 μ M Fura-2 AM (Thermo Fisher Scientific) and 0.01% Pluronic F127 (see above) for 15 min followed by 10 min for de-esterification.

    Techniques:

    Impact of mutations on the hydrogen peroxide-mediated TRPM2 activation. HEK293 cells were transiently transfected with an expression vector for EGFP and either human wild-type TRPM2 or a channel carrying the indicated mutation. At 24 h post transfection, cells were loaded with Fura2 and subjected to calcium imaging. At the indicated time, either buffer or hydrogen peroxide (final concentration 1 mM) was added. Analysis was restricted to EGFP-positive cells. For differentiation between responding and non-responding cells, the indicated threshold of 0.7 was chosen.

    Journal: Biochemical Journal

    Article Title: Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349

    doi: 10.1042/BCJ20170091

    Figure Lengend Snippet: Impact of mutations on the hydrogen peroxide-mediated TRPM2 activation. HEK293 cells were transiently transfected with an expression vector for EGFP and either human wild-type TRPM2 or a channel carrying the indicated mutation. At 24 h post transfection, cells were loaded with Fura2 and subjected to calcium imaging. At the indicated time, either buffer or hydrogen peroxide (final concentration 1 mM) was added. Analysis was restricted to EGFP-positive cells. For differentiation between responding and non-responding cells, the indicated threshold of 0.7 was chosen.

    Article Snippet: Ca2+ imaging One day post transfection, cells were loaded with Fura2 by adding Fura2/AM (Merck/Calbiochem, Darmstadt, Germany) to the medium to a final concentration of 4 µM and incubated for 30 min at 37°C/5% CO2 .

    Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Imaging, Concentration Assay

    Effects of nicardipine-containing media on STOCs and caffeine-induced responses. (a) Effects of nicardipine-containing external solutions on STOCs and I CAF at a V H of −20 mV. (b) Effects of nicardipine-containing external solutions on fura-2 microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while striped bars indicate application of nicardipine. Dotted lines indicate the zero current level.

    Journal: British Journal of Pharmacology

    Article Title: The properties of ryanodine-sensitive Ca2+ release in mouse gastric smooth muscle cells

    doi: 10.1038/sj.bjp.0704048

    Figure Lengend Snippet: Effects of nicardipine-containing media on STOCs and caffeine-induced responses. (a) Effects of nicardipine-containing external solutions on STOCs and I CAF at a V H of −20 mV. (b) Effects of nicardipine-containing external solutions on fura-2 microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while striped bars indicate application of nicardipine. Dotted lines indicate the zero current level.

    Article Snippet: Substances used for the experiments were nystatin (Nakarai Chemical, Japan), fura-2/AM (Dojindo, Japan), ionomycin (Sigma, U.S.A.), caffeine (Sigma, U.S.A.), carbachol (Sigma, U.S.A.), nicardipine (Sigma, U.S.A.), tetraethylammonium (Wako, Japan), iberiotoxin (Peptide Institute Inc., Japan), charybdotoxin (Peptide Institute Inc., Japan), apamin (Peptide Institute Inc., Japan), ryanodine (Wako, Japan), and dibutyryl cyclic AMP (Wako, Japan).

    Techniques:

    Effects of ryanodine on STOCs and caffeine-induced responses. (a) Effects of ryanodine on STOCs and I CAF at a V H of −20 mV. Dotted lines indicate the zero current level. (b) Effects of ryanodine on fura-2 microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed and open bars indicate application of caffeine and ryanodine.

    Journal: British Journal of Pharmacology

    Article Title: The properties of ryanodine-sensitive Ca2+ release in mouse gastric smooth muscle cells

    doi: 10.1038/sj.bjp.0704048

    Figure Lengend Snippet: Effects of ryanodine on STOCs and caffeine-induced responses. (a) Effects of ryanodine on STOCs and I CAF at a V H of −20 mV. Dotted lines indicate the zero current level. (b) Effects of ryanodine on fura-2 microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed and open bars indicate application of caffeine and ryanodine.

    Article Snippet: Substances used for the experiments were nystatin (Nakarai Chemical, Japan), fura-2/AM (Dojindo, Japan), ionomycin (Sigma, U.S.A.), caffeine (Sigma, U.S.A.), carbachol (Sigma, U.S.A.), nicardipine (Sigma, U.S.A.), tetraethylammonium (Wako, Japan), iberiotoxin (Peptide Institute Inc., Japan), charybdotoxin (Peptide Institute Inc., Japan), apamin (Peptide Institute Inc., Japan), ryanodine (Wako, Japan), and dibutyryl cyclic AMP (Wako, Japan).

    Techniques:

    Difference between the effects of caffeine and CCh. (a) Caffeine- and CCh-induced outward currents at a V H of −20 mV in gastric smooth muscle cells. (b) Caffeine- and CCh-induced [Ca 2+ ] i elevations measured with fura-2. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed and open bars indicate application of caffeine and CCh. (c) Effects of caffeine on the contracted gastric smooth muscle with CCh. Arrows indicate cumulative applications of CCh and caffeine and removal of the drugs.

    Journal: British Journal of Pharmacology

    Article Title: The properties of ryanodine-sensitive Ca2+ release in mouse gastric smooth muscle cells

    doi: 10.1038/sj.bjp.0704048

    Figure Lengend Snippet: Difference between the effects of caffeine and CCh. (a) Caffeine- and CCh-induced outward currents at a V H of −20 mV in gastric smooth muscle cells. (b) Caffeine- and CCh-induced [Ca 2+ ] i elevations measured with fura-2. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed and open bars indicate application of caffeine and CCh. (c) Effects of caffeine on the contracted gastric smooth muscle with CCh. Arrows indicate cumulative applications of CCh and caffeine and removal of the drugs.

    Article Snippet: Substances used for the experiments were nystatin (Nakarai Chemical, Japan), fura-2/AM (Dojindo, Japan), ionomycin (Sigma, U.S.A.), caffeine (Sigma, U.S.A.), carbachol (Sigma, U.S.A.), nicardipine (Sigma, U.S.A.), tetraethylammonium (Wako, Japan), iberiotoxin (Peptide Institute Inc., Japan), charybdotoxin (Peptide Institute Inc., Japan), apamin (Peptide Institute Inc., Japan), ryanodine (Wako, Japan), and dibutyryl cyclic AMP (Wako, Japan).

    Techniques:

    Effects of Ca 2+ -free media on STOCs and caffeine-induced responses. (a) Effects of nominally Ca 2+ -free external solutions on STOCs and I CAF at a V H of −20 mV. (b) Effects of nominally Ca 2+ -free external solutions on fura-2 microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while open bars indicate application of the Ca 2+ -free external solution. Dotted lines indicate the zero current level.

    Journal: British Journal of Pharmacology

    Article Title: The properties of ryanodine-sensitive Ca2+ release in mouse gastric smooth muscle cells

    doi: 10.1038/sj.bjp.0704048

    Figure Lengend Snippet: Effects of Ca 2+ -free media on STOCs and caffeine-induced responses. (a) Effects of nominally Ca 2+ -free external solutions on STOCs and I CAF at a V H of −20 mV. (b) Effects of nominally Ca 2+ -free external solutions on fura-2 microscopic [Ca 2+ ] i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while open bars indicate application of the Ca 2+ -free external solution. Dotted lines indicate the zero current level.

    Article Snippet: Substances used for the experiments were nystatin (Nakarai Chemical, Japan), fura-2/AM (Dojindo, Japan), ionomycin (Sigma, U.S.A.), caffeine (Sigma, U.S.A.), carbachol (Sigma, U.S.A.), nicardipine (Sigma, U.S.A.), tetraethylammonium (Wako, Japan), iberiotoxin (Peptide Institute Inc., Japan), charybdotoxin (Peptide Institute Inc., Japan), apamin (Peptide Institute Inc., Japan), ryanodine (Wako, Japan), and dibutyryl cyclic AMP (Wako, Japan).

    Techniques:

    IRBIT and Bcl2l10 cooperate to regulate IP 3 R activity. ( A ) Representative Ca 2+ response curve of Fura-2-loaded IRBIT KO MEF cells stimulated with 1 µM ATP at the indicated times. Cells were transfected with empty vector or with a plasmid expressing either FLAG-Bcl2l10 or FLAG-IRBIT alone, or FLAG-Bcl2l10 and FLAG-IRBIT together. Basal Fura-2 F 340 nm /F 380 nm = 0.61 ± 0.01, 0.59 ± 0.01, 0.59 ± 0.01 and 0.58 ± 0.01 for empty vector, Bcl2l10, IRBIT and Bcl2l10+IRBIT, respectively. ( B ) Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca 2+ peak (n: number of cells analyzed from five independent experiments). ( C ) Western blot of GST-pulldown performed with GST or GST-IP 3 BD on lysates of HeLa cells expressing HA-IRBIT alone or in combination with FLAG-Bcl2l10. Quantification was performed from three independent experiments. ( D ) Western blot of GST-pulldown performed with GST-IP 3 BD on recombinant Bcl2l10 alone or in combination with recombinant IRBIT produced in Sf9 cells. Quantification was performed from three independent experiments.( E ) Western blot of GST-pulldown performed with GST-IP 3 BD on lysates of HeLa cells expressing HA-IRBIT alone or in combination with FLAG-Bcl2l10 in the presence of 0, 1 or 10 µM IP 3 . Quantification was performed from three independent experiments. *p

    Journal: eLife

    Article Title: IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact

    doi: 10.7554/eLife.19896

    Figure Lengend Snippet: IRBIT and Bcl2l10 cooperate to regulate IP 3 R activity. ( A ) Representative Ca 2+ response curve of Fura-2-loaded IRBIT KO MEF cells stimulated with 1 µM ATP at the indicated times. Cells were transfected with empty vector or with a plasmid expressing either FLAG-Bcl2l10 or FLAG-IRBIT alone, or FLAG-Bcl2l10 and FLAG-IRBIT together. Basal Fura-2 F 340 nm /F 380 nm = 0.61 ± 0.01, 0.59 ± 0.01, 0.59 ± 0.01 and 0.58 ± 0.01 for empty vector, Bcl2l10, IRBIT and Bcl2l10+IRBIT, respectively. ( B ) Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca 2+ peak (n: number of cells analyzed from five independent experiments). ( C ) Western blot of GST-pulldown performed with GST or GST-IP 3 BD on lysates of HeLa cells expressing HA-IRBIT alone or in combination with FLAG-Bcl2l10. Quantification was performed from three independent experiments. ( D ) Western blot of GST-pulldown performed with GST-IP 3 BD on recombinant Bcl2l10 alone or in combination with recombinant IRBIT produced in Sf9 cells. Quantification was performed from three independent experiments.( E ) Western blot of GST-pulldown performed with GST-IP 3 BD on lysates of HeLa cells expressing HA-IRBIT alone or in combination with FLAG-Bcl2l10 in the presence of 0, 1 or 10 µM IP 3 . Quantification was performed from three independent experiments. *p

    Article Snippet: 24 hr after plasmid transfection or 48 hr after siRNA transfection, cells were loaded with 5 μM Fura-2 AM (DOJINDO) for 30 min, then placed on the stage of an inverted microscope (IX-70; Olympus, Japan) and perfused with balanced salt solution (BSS, 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 10 mM glucose, and 2 mM CaCl2 ).

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Recombinant, Produced

    Unphosphorylated IRBIT inhibits Bcl2l10 anti-apoptotic activity. ( A ) Bar graph showing the mean amplitude (±SEM) of the basal Fura-2 ratio value in MEF IRBIT KO cells before ATP stimulation (n: number of cells analyzed from three independent experiments). ( B ) Bar graph showing the mean amplitude (±SEM) of the basal Fura-2 ratio value in HeLa IRBIT KO cells before ATP stimulation (n: number of cells analyzed from three independent experiments). Insert: Western blot analysis of extracts of IRBIT KO HeLa cells transfected with control siRNA (Ctl) or with siRNA targeting bcl2l10 mRNA. ( C ) Representative fluorescence microscopy images of WT HeLa cells transfected with empty vector or expressing FLAG-Bcl2l10 and FLAG-IRBIT S68A alone or in combination and treated with 1 µM staurosporine (STS) for 4 hr or 20 µM tunicamycin (TUN) for 24 hr. Cells were stained with FLICA for active Caspase-3 (green) and Hoechst 33342 for nucleus labelling. DOI: http://dx.doi.org/10.7554/eLife.19896.009

    Journal: eLife

    Article Title: IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact

    doi: 10.7554/eLife.19896

    Figure Lengend Snippet: Unphosphorylated IRBIT inhibits Bcl2l10 anti-apoptotic activity. ( A ) Bar graph showing the mean amplitude (±SEM) of the basal Fura-2 ratio value in MEF IRBIT KO cells before ATP stimulation (n: number of cells analyzed from three independent experiments). ( B ) Bar graph showing the mean amplitude (±SEM) of the basal Fura-2 ratio value in HeLa IRBIT KO cells before ATP stimulation (n: number of cells analyzed from three independent experiments). Insert: Western blot analysis of extracts of IRBIT KO HeLa cells transfected with control siRNA (Ctl) or with siRNA targeting bcl2l10 mRNA. ( C ) Representative fluorescence microscopy images of WT HeLa cells transfected with empty vector or expressing FLAG-Bcl2l10 and FLAG-IRBIT S68A alone or in combination and treated with 1 µM staurosporine (STS) for 4 hr or 20 µM tunicamycin (TUN) for 24 hr. Cells were stained with FLICA for active Caspase-3 (green) and Hoechst 33342 for nucleus labelling. DOI: http://dx.doi.org/10.7554/eLife.19896.009

    Article Snippet: 24 hr after plasmid transfection or 48 hr after siRNA transfection, cells were loaded with 5 μM Fura-2 AM (DOJINDO) for 30 min, then placed on the stage of an inverted microscope (IX-70; Olympus, Japan) and perfused with balanced salt solution (BSS, 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 10 mM glucose, and 2 mM CaCl2 ).

    Techniques: Activity Assay, Western Blot, Transfection, CTL Assay, Fluorescence, Microscopy, Plasmid Preparation, Expressing, Staining

    IRBIT KO abolishes the effect of apoptosis-inducing drugs on Ca 2+ signaling. ( A ) Representative Ca 2+ response curve of Fura-2-loaded HeLa cells stimulated with 1 µM ATP at the indicated times. WT (left panel) or IRBIT KO (right panel) cells were treated with either DMSO (1/1000) for 4 hr, STS 1 µM for 30 min or TUN 20 µM for 4 hr before imaging. Basal Fura-2 F 340 nm /F 380 nm = 0.76 ± 0.01, 0.79 ± 0.01 and 0.75 ± 0.01 for WT cells treated with DMSO, STS and TUN, respectively; basal Fura-2 F 340 nm /F 380 nm = 0.75 ± 0.01, 0.78 ± 0.01 and 0.74 ± 0.01 for for IRBIT KO cells treated with DMSO, STS and TUN, respectively. ( B ) Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca 2+ peak (n: number of cells analyzed from three independent experiments). ( C ) Bar graph showing the mean relative amplitude (±SEM) of the ATP-induced Ca 2+ peak in cells treated with the indicated drugs compared to control (DMSO). ( D ) Representative images of Rhod-2-loaded HeLa cells treated with either DMSO (1/1000) for 8 hr, STS 1 µM for 90 min or TUN 20 µM for 8 hr. ( E ) Bar graph showing the mean relative Rhod-2 fluorescence intensity (±SEM) of HeLa cells treated with the indicated drugs compared to control (DMSO). (n: number of cells analyzed from three independent experiments.) *p

    Journal: eLife

    Article Title: IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact

    doi: 10.7554/eLife.19896

    Figure Lengend Snippet: IRBIT KO abolishes the effect of apoptosis-inducing drugs on Ca 2+ signaling. ( A ) Representative Ca 2+ response curve of Fura-2-loaded HeLa cells stimulated with 1 µM ATP at the indicated times. WT (left panel) or IRBIT KO (right panel) cells were treated with either DMSO (1/1000) for 4 hr, STS 1 µM for 30 min or TUN 20 µM for 4 hr before imaging. Basal Fura-2 F 340 nm /F 380 nm = 0.76 ± 0.01, 0.79 ± 0.01 and 0.75 ± 0.01 for WT cells treated with DMSO, STS and TUN, respectively; basal Fura-2 F 340 nm /F 380 nm = 0.75 ± 0.01, 0.78 ± 0.01 and 0.74 ± 0.01 for for IRBIT KO cells treated with DMSO, STS and TUN, respectively. ( B ) Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca 2+ peak (n: number of cells analyzed from three independent experiments). ( C ) Bar graph showing the mean relative amplitude (±SEM) of the ATP-induced Ca 2+ peak in cells treated with the indicated drugs compared to control (DMSO). ( D ) Representative images of Rhod-2-loaded HeLa cells treated with either DMSO (1/1000) for 8 hr, STS 1 µM for 90 min or TUN 20 µM for 8 hr. ( E ) Bar graph showing the mean relative Rhod-2 fluorescence intensity (±SEM) of HeLa cells treated with the indicated drugs compared to control (DMSO). (n: number of cells analyzed from three independent experiments.) *p

    Article Snippet: 24 hr after plasmid transfection or 48 hr after siRNA transfection, cells were loaded with 5 μM Fura-2 AM (DOJINDO) for 30 min, then placed on the stage of an inverted microscope (IX-70; Olympus, Japan) and perfused with balanced salt solution (BSS, 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 10 mM glucose, and 2 mM CaCl2 ).

    Techniques: Imaging, Fluorescence

    Bcl2l10 interacts with and regulates IP 3 R. ( A ) Clustal Omega alignment of human Bcl2l10 and zebrafish Nrz primary structures. Identical residues are boxed in blue. The positions of conserved Bcl-2 homology (BH) domains and of the C-terminal transmembrane (TM) domain are indicated. ( B ) Western blot of immunoprecipitation (IP) between the three endogenous IP 3 R isoforms (IP 3 R1, IP 3 R2 and IP 3 R3) and endogenous Bcl2l10. IgG antibodies are indicated by arrows. Arrow heads indicate bands corresponding to Bcl2l10 in IP. Western blots are representative of three independent experiments. ( C ) Western blot of GST-pulldown performed with GST, GST-IP 3 R∆CD or GST-IP 3 BD on lysates of HeLa cells expressing FLAG-Bcl2l10 or FLAG-∆BH4Bcl2l10. Arrows indicate the bands corresponding to GST, GST-IP 3 R∆CD and GST-IP 3 BD. Western blot representative of three independents experiments. ( D ) Left panel: representative Ca 2+ response curve of Fura-2-loaded cells stimulated with 1 µM ATP at the indicated times. Cells were transfected with empty vector or FLAG-Bcl2l10. Basal Fura-2 F 340 nm /F 380 nm = 0.61 ± 0.01 and 0.59 ± 0.01 for empty vector and Bcl2l10 cells, respectively. Right panel: Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca 2+ peak (n: number of cells analyzed from five independent experiments). ( E ) Left panel: representative response curve of Fura-2-loaded cells treated with 1 µM thapsigargin at the indicated times. Right panel: Bar graph showing the mean area under curve (AUC) (±SEM) of the thapsigargin-induced Ca 2+ peak (n: number of cells analyzed from three independent experiments). ***p

    Journal: eLife

    Article Title: IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact

    doi: 10.7554/eLife.19896

    Figure Lengend Snippet: Bcl2l10 interacts with and regulates IP 3 R. ( A ) Clustal Omega alignment of human Bcl2l10 and zebrafish Nrz primary structures. Identical residues are boxed in blue. The positions of conserved Bcl-2 homology (BH) domains and of the C-terminal transmembrane (TM) domain are indicated. ( B ) Western blot of immunoprecipitation (IP) between the three endogenous IP 3 R isoforms (IP 3 R1, IP 3 R2 and IP 3 R3) and endogenous Bcl2l10. IgG antibodies are indicated by arrows. Arrow heads indicate bands corresponding to Bcl2l10 in IP. Western blots are representative of three independent experiments. ( C ) Western blot of GST-pulldown performed with GST, GST-IP 3 R∆CD or GST-IP 3 BD on lysates of HeLa cells expressing FLAG-Bcl2l10 or FLAG-∆BH4Bcl2l10. Arrows indicate the bands corresponding to GST, GST-IP 3 R∆CD and GST-IP 3 BD. Western blot representative of three independents experiments. ( D ) Left panel: representative Ca 2+ response curve of Fura-2-loaded cells stimulated with 1 µM ATP at the indicated times. Cells were transfected with empty vector or FLAG-Bcl2l10. Basal Fura-2 F 340 nm /F 380 nm = 0.61 ± 0.01 and 0.59 ± 0.01 for empty vector and Bcl2l10 cells, respectively. Right panel: Bar graph showing the mean amplitude (±SEM) of the ATP-induced Ca 2+ peak (n: number of cells analyzed from five independent experiments). ( E ) Left panel: representative response curve of Fura-2-loaded cells treated with 1 µM thapsigargin at the indicated times. Right panel: Bar graph showing the mean area under curve (AUC) (±SEM) of the thapsigargin-induced Ca 2+ peak (n: number of cells analyzed from three independent experiments). ***p

    Article Snippet: 24 hr after plasmid transfection or 48 hr after siRNA transfection, cells were loaded with 5 μM Fura-2 AM (DOJINDO) for 30 min, then placed on the stage of an inverted microscope (IX-70; Olympus, Japan) and perfused with balanced salt solution (BSS, 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 10 mM glucose, and 2 mM CaCl2 ).

    Techniques: Western Blot, Immunoprecipitation, Expressing, Transfection, Plasmid Preparation

    IRBIT KO reduces ER-mitochondria contact in MEF cells. ( A ) Bar graph showing the mean amplitude (±SEM) of the basal Fura-2 ratio value in MEF cells before ATP stimulation (n: number of cells analyzed from three independent experiments). ( B ) Left panel: immunofluorescence of WT and IRBIT KO MEF cells transfected with an ER marker (KDEL-GFP (green)) and stained with anti-TOM20 antibody (magenta) for mitochondria labelling. Right panel: histogram showing the mean Mander’s overlap coefficient (±SEM) and the mean Pearson coefficient (±SEM) of WT and IRBIT KO MEF cells (n = three independent experiments, ~20 cells analyzed per condition for each experiment). DOI: http://dx.doi.org/10.7554/eLife.19896.011

    Journal: eLife

    Article Title: IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact

    doi: 10.7554/eLife.19896

    Figure Lengend Snippet: IRBIT KO reduces ER-mitochondria contact in MEF cells. ( A ) Bar graph showing the mean amplitude (±SEM) of the basal Fura-2 ratio value in MEF cells before ATP stimulation (n: number of cells analyzed from three independent experiments). ( B ) Left panel: immunofluorescence of WT and IRBIT KO MEF cells transfected with an ER marker (KDEL-GFP (green)) and stained with anti-TOM20 antibody (magenta) for mitochondria labelling. Right panel: histogram showing the mean Mander’s overlap coefficient (±SEM) and the mean Pearson coefficient (±SEM) of WT and IRBIT KO MEF cells (n = three independent experiments, ~20 cells analyzed per condition for each experiment). DOI: http://dx.doi.org/10.7554/eLife.19896.011

    Article Snippet: 24 hr after plasmid transfection or 48 hr after siRNA transfection, cells were loaded with 5 μM Fura-2 AM (DOJINDO) for 30 min, then placed on the stage of an inverted microscope (IX-70; Olympus, Japan) and perfused with balanced salt solution (BSS, 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 10 mM glucose, and 2 mM CaCl2 ).

    Techniques: Immunofluorescence, Transfection, Marker, Staining

    Cellular stress from AS aggregates causes early reduction in cytosolic calcium followed by later increase Cytosolic Ca 2+ levels were quantified by the Ca 2+ sensor Fura‐2 and converted to absolute concentrations using the Fura‐2 Calcium Imaging Calibration Kit. The AS aggregation inhibitor ASI‐1D (20 μM) was used to validate that phenotypes were aggregate‐dependent. Statistical analyses were performed using one‐way ANOVA multiple comparisons with Sidak post hoc test. Mitotic OLN‐t40‐AS cells were transfected with p25α and the fluorescent transfection marker tdTomato. OLN‐t40‐AS cells transfected with tdTomato and empty expression vectors served as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0001, ** P = 0.0002, # P = 0.0011, ## P = 0.0033). The average Ca 2+ level of individual experiments was calculated by measuring > 50 or more tdTomato expressing cells. Non‐mitotic SH‐SY5Y cells were generated by treatment with retinoic acid (RA; 10 μM) for 2 days, after which AS expression was induced by removal of doxycycline (dox) and cytosolic Ca 2+ measured after 5 days and 10 days of AS expression. See timeline under bars. Cells induced to express β‐galactosidase (b‐gal) upon dox removal were used as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 4 (* P = 0.0005, ** P = 0.0005, # P = 0.0062, ## P = 0.0055). The average Ca 2+ level was calculated by measuring > 200 randomly selected cells in each experiment. Primary hippocampal neurons were isolated from new‐born (P0) mice expressing human AS under the mThy1 promoter and wild‐type (wt) littermates. Cytosolic Ca 2+ was measured after 5 days in vitro (5 DIV) and 14 days in vitro culture (14 DIV). See timeline under graphs. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.002, ** P = 0.0007, # P = 0.0071, ## P = 0.0455). The average cellular Ca 2+ is based on > 500 neurons per experiment. Cytosolic Ca 2+ in SHSY5Y cells as in (B) measured every second day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0377, ** P = 0.0057, *** P = 0.03, # P = 0.045, ## P = 0.0229). Cytosolic Ca 2+ in primary hippocampal neurons as in (C) measured every third day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.01, ** P = 0.0073, # P = 0.0058).

    Journal: EMBO Reports

    Article Title: Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

    doi: 10.15252/embr.201744617

    Figure Lengend Snippet: Cellular stress from AS aggregates causes early reduction in cytosolic calcium followed by later increase Cytosolic Ca 2+ levels were quantified by the Ca 2+ sensor Fura‐2 and converted to absolute concentrations using the Fura‐2 Calcium Imaging Calibration Kit. The AS aggregation inhibitor ASI‐1D (20 μM) was used to validate that phenotypes were aggregate‐dependent. Statistical analyses were performed using one‐way ANOVA multiple comparisons with Sidak post hoc test. Mitotic OLN‐t40‐AS cells were transfected with p25α and the fluorescent transfection marker tdTomato. OLN‐t40‐AS cells transfected with tdTomato and empty expression vectors served as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0001, ** P = 0.0002, # P = 0.0011, ## P = 0.0033). The average Ca 2+ level of individual experiments was calculated by measuring > 50 or more tdTomato expressing cells. Non‐mitotic SH‐SY5Y cells were generated by treatment with retinoic acid (RA; 10 μM) for 2 days, after which AS expression was induced by removal of doxycycline (dox) and cytosolic Ca 2+ measured after 5 days and 10 days of AS expression. See timeline under bars. Cells induced to express β‐galactosidase (b‐gal) upon dox removal were used as negative controls. Bars display Ca 2+ concentrations as mean ± SD, N = 4 (* P = 0.0005, ** P = 0.0005, # P = 0.0062, ## P = 0.0055). The average Ca 2+ level was calculated by measuring > 200 randomly selected cells in each experiment. Primary hippocampal neurons were isolated from new‐born (P0) mice expressing human AS under the mThy1 promoter and wild‐type (wt) littermates. Cytosolic Ca 2+ was measured after 5 days in vitro (5 DIV) and 14 days in vitro culture (14 DIV). See timeline under graphs. Bars display Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.002, ** P = 0.0007, # P = 0.0071, ## P = 0.0455). The average cellular Ca 2+ is based on > 500 neurons per experiment. Cytosolic Ca 2+ in SHSY5Y cells as in (B) measured every second day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.0377, ** P = 0.0057, *** P = 0.03, # P = 0.045, ## P = 0.0229). Cytosolic Ca 2+ in primary hippocampal neurons as in (C) measured every third day. Points represent Ca 2+ concentrations as mean ± SD, N = 3 (* P = 0.01, ** P = 0.0073, # P = 0.0058).

    Article Snippet: The Fura‐2 ratios were converted to molar concentration using Fura‐2 Calcium Imaging Calibration Kit (Molecular Probes, F6774), generating a standard curve from 0 to 350 nM Ca2+ .

    Techniques: Imaging, Transfection, Marker, Expressing, Generated, Isolation, Mouse Assay, In Vitro

    Allosteric activation of SERCA mimics the Ca 2+ dysregulation of aggregated AS. CPA reduces NF‐κB nuclear translocation in OLN cells and increases survival in neurons Allosteric activation of SERCA by XCT790 mimics the early decrease and later increase in Ca 2+ observed upon intracellular AS aggregation. Cytosolic Ca 2+ levels were quantified by the Ca 2+ sensor Fura‐2 and converted to absolute values using the Fura‐2 Calcium Imaging Calibration Kit. Non‐mitotic SH‐SY5Y wt cells were generated by treatment with 10 μM retinoic acid for 2 days before treatment with SERCA activator, 10 μM XCT790 (Sigma‐Aldrich). The cytosolic Ca 2+ in SH‐SY5Y cells was measured after 4, 24 and 48 h. Points represent Ca 2+ concentrations as mean ± SD, N = 2. . Bars represent the average percentage of transfected cells with NF‐κB translocated to the nucleus ± SD in > 100 transfected cells in each experiment (one‐way ANOVA multiple comparisons with Sidak post hoc test, * P = 0.0001 and ** P = 0.0006). N = 3. Survival of neurons from day 6 to day 14 was quantified by counting MAP2‐positive cells. Representative MAP2 staining pattern (green) of primary hippocampal neurons with NeuN marking neuronal nuclei. Scale bar is 50 μm. Bars represent remaining MAP‐2‐positive neurons at day 14 normalised to the number present at day 6, presented as means of three individual experiments ± SD each of > 300 neurons (one‐way ANOVA multiple comparisons with Sidak post hoc test, * P = 0.0003, ** P = 0.0001).

    Journal: EMBO Reports

    Article Title: Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

    doi: 10.15252/embr.201744617

    Figure Lengend Snippet: Allosteric activation of SERCA mimics the Ca 2+ dysregulation of aggregated AS. CPA reduces NF‐κB nuclear translocation in OLN cells and increases survival in neurons Allosteric activation of SERCA by XCT790 mimics the early decrease and later increase in Ca 2+ observed upon intracellular AS aggregation. Cytosolic Ca 2+ levels were quantified by the Ca 2+ sensor Fura‐2 and converted to absolute values using the Fura‐2 Calcium Imaging Calibration Kit. Non‐mitotic SH‐SY5Y wt cells were generated by treatment with 10 μM retinoic acid for 2 days before treatment with SERCA activator, 10 μM XCT790 (Sigma‐Aldrich). The cytosolic Ca 2+ in SH‐SY5Y cells was measured after 4, 24 and 48 h. Points represent Ca 2+ concentrations as mean ± SD, N = 2. . Bars represent the average percentage of transfected cells with NF‐κB translocated to the nucleus ± SD in > 100 transfected cells in each experiment (one‐way ANOVA multiple comparisons with Sidak post hoc test, * P = 0.0001 and ** P = 0.0006). N = 3. Survival of neurons from day 6 to day 14 was quantified by counting MAP2‐positive cells. Representative MAP2 staining pattern (green) of primary hippocampal neurons with NeuN marking neuronal nuclei. Scale bar is 50 μm. Bars represent remaining MAP‐2‐positive neurons at day 14 normalised to the number present at day 6, presented as means of three individual experiments ± SD each of > 300 neurons (one‐way ANOVA multiple comparisons with Sidak post hoc test, * P = 0.0003, ** P = 0.0001).

    Article Snippet: The Fura‐2 ratios were converted to molar concentration using Fura‐2 Calcium Imaging Calibration Kit (Molecular Probes, F6774), generating a standard curve from 0 to 350 nM Ca2+ .

    Techniques: Activation Assay, Translocation Assay, Imaging, Generated, Transfection, Staining

    Emission spectra of RES and Fura-2. Separate measurements (A) of 340 nm (Ca 2+ -bound, blue) and 380 nm (Ca 2+ -free, orange) signal intensity in MDA cells with 5 µM TG and 100 µM RES treatments at 1 min and 6 min, respectively, as indicated by the arrows. This data (A) is representative of all experiments conducted at these concentrations. Emission spectra (B) in 1 mM Ca 2+ solution when excited at 340 nm of 100 µM RES (blue), and 5 µM Fura-2 (yellow).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: Emission spectra of RES and Fura-2. Separate measurements (A) of 340 nm (Ca 2+ -bound, blue) and 380 nm (Ca 2+ -free, orange) signal intensity in MDA cells with 5 µM TG and 100 µM RES treatments at 1 min and 6 min, respectively, as indicated by the arrows. This data (A) is representative of all experiments conducted at these concentrations. Emission spectra (B) in 1 mM Ca 2+ solution when excited at 340 nm of 100 µM RES (blue), and 5 µM Fura-2 (yellow).

    Article Snippet: Cells were imaged following Fura-2 loading with an Olympus IX51 inverted microscope.

    Techniques: Multiple Displacement Amplification

    PMCA inhibition by RES is unique among polyphenols. Structures of resveratrol (A), quercetin (B), and epigallocatechin gallate (EGCG) (C). PDF cells (D) were imaged for 15 min using Fura-2. Arrows represent treatment with 5 µM TG, and either 100 µM RES (black), 100 µM quercetin (blue), 100 µM EGCG (red) or vehicle (green) at 1 min and 6 min, respectively. Quercetin, EGCG and vehicle all showed statistically significant differences in [Ca 2+ ] i changes from RES in PDF cells (* signifies a statistically significant difference from RES at p

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: PMCA inhibition by RES is unique among polyphenols. Structures of resveratrol (A), quercetin (B), and epigallocatechin gallate (EGCG) (C). PDF cells (D) were imaged for 15 min using Fura-2. Arrows represent treatment with 5 µM TG, and either 100 µM RES (black), 100 µM quercetin (blue), 100 µM EGCG (red) or vehicle (green) at 1 min and 6 min, respectively. Quercetin, EGCG and vehicle all showed statistically significant differences in [Ca 2+ ] i changes from RES in PDF cells (* signifies a statistically significant difference from RES at p

    Article Snippet: Cells were imaged following Fura-2 loading with an Olympus IX51 inverted microscope.

    Techniques: Inhibition

    The direct effects of RES on Fura-2 fluorescence. Solutions containing 5 µM Fura-2 pentasodium salt were titrated with RES at 0 µM (blue), 1 µM (orange), 10 µM (gray), 25 µM (yellow), 50 µM (red), 75 µM (green), and 100 µM (purple). The samples were excited at 340 nm and 380 nm and the emission of each sample was measured at 510 nm. Each measurement was performed in triplicate in PBS with 0 mM Ca 2+ or 1 mM Ca 2+ as outlined in the figure (*signifies a statistically significant difference from Fura-2 emission at 510 nm at p

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: The direct effects of RES on Fura-2 fluorescence. Solutions containing 5 µM Fura-2 pentasodium salt were titrated with RES at 0 µM (blue), 1 µM (orange), 10 µM (gray), 25 µM (yellow), 50 µM (red), 75 µM (green), and 100 µM (purple). The samples were excited at 340 nm and 380 nm and the emission of each sample was measured at 510 nm. Each measurement was performed in triplicate in PBS with 0 mM Ca 2+ or 1 mM Ca 2+ as outlined in the figure (*signifies a statistically significant difference from Fura-2 emission at 510 nm at p

    Article Snippet: Cells were imaged following Fura-2 loading with an Olympus IX51 inverted microscope.

    Techniques: Fluorescence

    PMCA activity is inhibited by RES. Cells were monitored for changes in [Ca 2+ ] i for 15 min using Fura-2. Graphs depict changes in [Ca 2+ ] i as measured by 340 nm/380 nm ratio signal intensity. PMCA inhibition with LaCl 3 (A) (arrows mark addition of 5 µM TG and 1 mM LaCl 3 at 1 min and 6 min, respectively). LaCl 3 induced an increase in [Ca 2+ ] i in PDF (blue) and MDA (black) cell types as compared to vehicle only treatment in the respective cell types (PDF cells represented by green, MDA cells represented by red), indicative of PMCA inhibition. To ensure there were no calcium independent effects, BAPTA and Fura-2 were co-loaded into cells (B) (arrows mark addition of 5 µM TG and 50 µM RES at 1 min and 6 min, respectively). PMCA inhibition by RES (C), (arrows mark addition of 5 µM TG and 100 µM RES at 1 min and 6 min, respectively) in both PDF (blue) and MDA (black) cells. RES induced a statistically significant rise in [Ca 2+ ] i as compared to a vehicle only treatment (* signifies a statistically significant difference from respective vehicle controls at p

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: PMCA activity is inhibited by RES. Cells were monitored for changes in [Ca 2+ ] i for 15 min using Fura-2. Graphs depict changes in [Ca 2+ ] i as measured by 340 nm/380 nm ratio signal intensity. PMCA inhibition with LaCl 3 (A) (arrows mark addition of 5 µM TG and 1 mM LaCl 3 at 1 min and 6 min, respectively). LaCl 3 induced an increase in [Ca 2+ ] i in PDF (blue) and MDA (black) cell types as compared to vehicle only treatment in the respective cell types (PDF cells represented by green, MDA cells represented by red), indicative of PMCA inhibition. To ensure there were no calcium independent effects, BAPTA and Fura-2 were co-loaded into cells (B) (arrows mark addition of 5 µM TG and 50 µM RES at 1 min and 6 min, respectively). PMCA inhibition by RES (C), (arrows mark addition of 5 µM TG and 100 µM RES at 1 min and 6 min, respectively) in both PDF (blue) and MDA (black) cells. RES induced a statistically significant rise in [Ca 2+ ] i as compared to a vehicle only treatment (* signifies a statistically significant difference from respective vehicle controls at p

    Article Snippet: Cells were imaged following Fura-2 loading with an Olympus IX51 inverted microscope.

    Techniques: Activity Assay, Inhibition, Multiple Displacement Amplification

    Overexpressed wtTG2 appears in the mitochondria and induces enhanced mitochondrial Ca 2+ accumulation. (A) Time dependent changes in the mitochondrial Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Rhod-2/AM. (B) Time dependent changes in the cytosolic Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Fura-2/AM. (C) Time dependent changes in the mitochondrial and cytoplasmic TG2 expressions of Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Western blot analysis. Mitochondrial HSP60 and cytoplasmic β-actin were used as loading controls; while β-tubulin was used to check for cytoplasmic contamination of mitochondria. (D) Time dependent changes in the mitochondrial and cytoplasmic TG2 activities of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment. (E) Confocal images taken at 16 h following Dox treatment show Tet-on wtTG2 cells expressing TG2 (green) colocalized with the mitochondrial calcium indicator Rhod-2/AM (red). Scale bar = 5 µM. All the data presented represent mean±S.D. of at least three determinations. Significantly different from the TG2C277S cell line detected at the same time point (* P

    Journal: PLoS ONE

    Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

    doi: 10.1371/journal.pone.0081516

    Figure Lengend Snippet: Overexpressed wtTG2 appears in the mitochondria and induces enhanced mitochondrial Ca 2+ accumulation. (A) Time dependent changes in the mitochondrial Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Rhod-2/AM. (B) Time dependent changes in the cytosolic Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Fura-2/AM. (C) Time dependent changes in the mitochondrial and cytoplasmic TG2 expressions of Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Western blot analysis. Mitochondrial HSP60 and cytoplasmic β-actin were used as loading controls; while β-tubulin was used to check for cytoplasmic contamination of mitochondria. (D) Time dependent changes in the mitochondrial and cytoplasmic TG2 activities of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment. (E) Confocal images taken at 16 h following Dox treatment show Tet-on wtTG2 cells expressing TG2 (green) colocalized with the mitochondrial calcium indicator Rhod-2/AM (red). Scale bar = 5 µM. All the data presented represent mean±S.D. of at least three determinations. Significantly different from the TG2C277S cell line detected at the same time point (* P

    Article Snippet: [Ca2+ ]ER determination with Mag-Fura-2 AM Endoplasmic reticulum calcium level, [Ca2+ ]ER , was measured using Mag-Fura-2 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Plasmid Preparation, Western Blot, Expressing

    Transglutaminase 2 affects both Ins 3 P and ryanodine sensitive receptors to enhance mitochondrial Ca 2+ uptake. (A–C) Representative recordings of thapsigargin-induced intra-mitochondrial Ca 2+ elevations in Tet-on cells treated previously with Dox (50 µM) for 18 h, in the presence or absence of 1 µM TMB-8, an Ins 3 P receptor antagonist, 1 µM ryanodine, or both. Tg response of Tet-on vector and Tet-on TG2C277S cells treated with Dox (50 µM) for 18 h is also shown. (D–F) Representative recordings of tg-induced intra-mitochondrial Ca 2+ elevations in in WT MEF and TG2 KO MEF cells in the presence or absence of 1 µM TMB-8, 1 µM Ryanodine or both. Top panels , Representative kinetic average changes in mitochondrial Ca 2+ signals induced by tg (5 µM) over time are shown. Bottom panels , Areas, statistical evaluation of integrated Ca 2+ responses are shown. AUC, area under the curve. (G) Resting value of Mag-Fura 2 ratios (340/380) were measured in Mag-fura-2/AM loaded WT MEF and TG2 KO MEF cells. All the presented data are representative of at least three experiments and are shown as mean ± SD. *, P

    Journal: PLoS ONE

    Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

    doi: 10.1371/journal.pone.0081516

    Figure Lengend Snippet: Transglutaminase 2 affects both Ins 3 P and ryanodine sensitive receptors to enhance mitochondrial Ca 2+ uptake. (A–C) Representative recordings of thapsigargin-induced intra-mitochondrial Ca 2+ elevations in Tet-on cells treated previously with Dox (50 µM) for 18 h, in the presence or absence of 1 µM TMB-8, an Ins 3 P receptor antagonist, 1 µM ryanodine, or both. Tg response of Tet-on vector and Tet-on TG2C277S cells treated with Dox (50 µM) for 18 h is also shown. (D–F) Representative recordings of tg-induced intra-mitochondrial Ca 2+ elevations in in WT MEF and TG2 KO MEF cells in the presence or absence of 1 µM TMB-8, 1 µM Ryanodine or both. Top panels , Representative kinetic average changes in mitochondrial Ca 2+ signals induced by tg (5 µM) over time are shown. Bottom panels , Areas, statistical evaluation of integrated Ca 2+ responses are shown. AUC, area under the curve. (G) Resting value of Mag-Fura 2 ratios (340/380) were measured in Mag-fura-2/AM loaded WT MEF and TG2 KO MEF cells. All the presented data are representative of at least three experiments and are shown as mean ± SD. *, P

    Article Snippet: [Ca2+ ]ER determination with Mag-Fura-2 AM Endoplasmic reticulum calcium level, [Ca2+ ]ER , was measured using Mag-Fura-2 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Plasmid Preparation

    RAP1GDS1 mediates the enhancing effect of TG2 on the calcium release from ER and on the subsequent mitochondrial Ca 2+ uptake. (A) RAP1GDS1 and TG2 protein expression levels detected by Western blotting in Tet-on wtTG2, Tet-on wtTG2 with sh vector and Tet-on wtTG2 with shRAP1GDS1 (sh1) cells following 18 hours Dox (50 µM) treatment. β-actin was used as loading control. Tet-On wtTG2 with sh vector and sh1 cells treated with Dox for 18 hours were exposed to 5 µM thapsigargin. (B) A representative recording of the tg-induced Ca 2+ release from the ER recorded by Mag-Fura-2/AM fluorescence is shown. Right panel , statistical evaluation of the tg-induced ER Ca 2+ depletion. (C) A representative recording of the tg-induced intra-mitochondrial Ca 2+ changes is shown. Right panel , Area, statistical evaluation of integrated Ca 2+ response. AUC, area under the curve. These data are representative of at least three experiments and shown as mean ± SD. *, P

    Journal: PLoS ONE

    Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

    doi: 10.1371/journal.pone.0081516

    Figure Lengend Snippet: RAP1GDS1 mediates the enhancing effect of TG2 on the calcium release from ER and on the subsequent mitochondrial Ca 2+ uptake. (A) RAP1GDS1 and TG2 protein expression levels detected by Western blotting in Tet-on wtTG2, Tet-on wtTG2 with sh vector and Tet-on wtTG2 with shRAP1GDS1 (sh1) cells following 18 hours Dox (50 µM) treatment. β-actin was used as loading control. Tet-On wtTG2 with sh vector and sh1 cells treated with Dox for 18 hours were exposed to 5 µM thapsigargin. (B) A representative recording of the tg-induced Ca 2+ release from the ER recorded by Mag-Fura-2/AM fluorescence is shown. Right panel , statistical evaluation of the tg-induced ER Ca 2+ depletion. (C) A representative recording of the tg-induced intra-mitochondrial Ca 2+ changes is shown. Right panel , Area, statistical evaluation of integrated Ca 2+ response. AUC, area under the curve. These data are representative of at least three experiments and shown as mean ± SD. *, P

    Article Snippet: [Ca2+ ]ER determination with Mag-Fura-2 AM Endoplasmic reticulum calcium level, [Ca2+ ]ER , was measured using Mag-Fura-2 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Fluorescence

    Brain slices were incubated in a stage top chamber with 5%CO2 at 37°C. Fura-2 images were obtained for brain slices at 5s intervals for a total of 15min. Time lapse series of P4 GFP-expressing OPCs in the Dorsolateral subventricular zone ( SVZ ) ( A ) and in the Corpus Callosum ( CC ) ( B ). Each frame represents a single section of a Fura-2 time lapse experiment. An increased Fura-2 fluorescence ratio is indicated by warmer colors. Time is denoted in minutes in the upper right corner and the area of the CC and SVZ is indicated in the inset. LV : lateral ventricle. Scale bar ( A ) 100μm, ( B ) 50μm. ( C and D ) VOCC activity was examined in P4 GFP-expressing OPCs from the SVZ area and OLs from the CC. Note that each trace corresponds to a single cell and the time of addition of external solution containing high K + is indicated by the horizontal bars. ( E ) K + -induced Ca ++ uptake in P4 OPCs from the SVZ was abolished in 25μM verapamil, 25μM nifedipine and in the absence of external Ca ++ (-Ca ++ ). The graph show the average amplitude calculated from the responding cells, expressed as percentage of change of the emission intensities. ( F ) VOCC activity was examined in P4 and P8 GFP-expressing OPCs from the SVZ area and OLs from the CC. The graph show the average maximal peak values and plateau values during minutes 9-11, calculated from the responding cells, expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Multiple kinase pathways regulate voltage-dependent Ca++ influx and migration in oligodendrocyte precursor cells

    doi: 10.1523/JNEUROSCI.5086-09.2010

    Figure Lengend Snippet: Brain slices were incubated in a stage top chamber with 5%CO2 at 37°C. Fura-2 images were obtained for brain slices at 5s intervals for a total of 15min. Time lapse series of P4 GFP-expressing OPCs in the Dorsolateral subventricular zone ( SVZ ) ( A ) and in the Corpus Callosum ( CC ) ( B ). Each frame represents a single section of a Fura-2 time lapse experiment. An increased Fura-2 fluorescence ratio is indicated by warmer colors. Time is denoted in minutes in the upper right corner and the area of the CC and SVZ is indicated in the inset. LV : lateral ventricle. Scale bar ( A ) 100μm, ( B ) 50μm. ( C and D ) VOCC activity was examined in P4 GFP-expressing OPCs from the SVZ area and OLs from the CC. Note that each trace corresponds to a single cell and the time of addition of external solution containing high K + is indicated by the horizontal bars. ( E ) K + -induced Ca ++ uptake in P4 OPCs from the SVZ was abolished in 25μM verapamil, 25μM nifedipine and in the absence of external Ca ++ (-Ca ++ ). The graph show the average amplitude calculated from the responding cells, expressed as percentage of change of the emission intensities. ( F ) VOCC activity was examined in P4 and P8 GFP-expressing OPCs from the SVZ area and OLs from the CC. The graph show the average maximal peak values and plateau values during minutes 9-11, calculated from the responding cells, expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). *p

    Article Snippet: The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments, Novato, CA).

    Techniques: Incubation, Expressing, Fluorescence, Activity Assay

    ( A ) Effect of different concentrations of PDGF on Ca ++ influx induced by high K + in OPCs. The graph show the average maximal peak values and plateau values in the presence of different PDGF concentrations. ( B ) Fura-2 imaging of Ca ++ response to 20mM K + in selected OPCs from control and treated cultures with the TKr inhibitor AG-1296 (1μM) applied two minutes before high K + . ( C ) OPCs were treated with AG-1296 (1μM) or genistein (10μM) in the presence of 20ng/ml or 40ng/ml PDGF. The graph show the amplitude of Ca ++ uptake (peak) calculated from the responding cells. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). **p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Multiple kinase pathways regulate voltage-dependent Ca++ influx and migration in oligodendrocyte precursor cells

    doi: 10.1523/JNEUROSCI.5086-09.2010

    Figure Lengend Snippet: ( A ) Effect of different concentrations of PDGF on Ca ++ influx induced by high K + in OPCs. The graph show the average maximal peak values and plateau values in the presence of different PDGF concentrations. ( B ) Fura-2 imaging of Ca ++ response to 20mM K + in selected OPCs from control and treated cultures with the TKr inhibitor AG-1296 (1μM) applied two minutes before high K + . ( C ) OPCs were treated with AG-1296 (1μM) or genistein (10μM) in the presence of 20ng/ml or 40ng/ml PDGF. The graph show the amplitude of Ca ++ uptake (peak) calculated from the responding cells. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). **p

    Article Snippet: The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments, Novato, CA).

    Techniques: Imaging

    ( A ) Fura-2 imaging of Ca ++ response to 20mM K + in control OPCs (Basal). Effect of PMA (10μM) ( B ) and Chelerythrine (50μM) ( C ) on Ca ++ influx induced by high K + in OPCs. As indicated by the horizontal bars, the phorbol ester PMA and the PKC inhibitor Chelerythrine were applied two minutes before high K + stimulation. ( D ) The PKC activator PMA (10μM) was applied after K + stimulation. ( E ) The graph show the average maximal peak values and plateau values (minutes 5-7) for each experimental condition, calculated from the responding cells and expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). **p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Multiple kinase pathways regulate voltage-dependent Ca++ influx and migration in oligodendrocyte precursor cells

    doi: 10.1523/JNEUROSCI.5086-09.2010

    Figure Lengend Snippet: ( A ) Fura-2 imaging of Ca ++ response to 20mM K + in control OPCs (Basal). Effect of PMA (10μM) ( B ) and Chelerythrine (50μM) ( C ) on Ca ++ influx induced by high K + in OPCs. As indicated by the horizontal bars, the phorbol ester PMA and the PKC inhibitor Chelerythrine were applied two minutes before high K + stimulation. ( D ) The PKC activator PMA (10μM) was applied after K + stimulation. ( E ) The graph show the average maximal peak values and plateau values (minutes 5-7) for each experimental condition, calculated from the responding cells and expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). **p

    Article Snippet: The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments, Novato, CA).

    Techniques: Imaging

    ( A ) Fura-2 imaging of Ca ++ responses to 20mM K + in primary cultures of OPCs. The time of addition of high K + containing external solution is indicated by the horizontal bar. Note that each trace corresponds to a single cell. ( B ) K + -induced Ca ++ uptake was increased in 5μM BayK and abolished in 10μM Cd ++ , 25μM verapamil, 25μM nifedipine and in the absence of external Ca ++ (-Ca ++ ). The graph shows the average amplitude (peak) calculated from the responding cells, expressed as percentage of change of the emission intensities. Each agonist was applied by a fast and local perfusion system. Values are expressed as mean ± SEM of at least four independent experiments ( n > 500 cells for each condition). **p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Multiple kinase pathways regulate voltage-dependent Ca++ influx and migration in oligodendrocyte precursor cells

    doi: 10.1523/JNEUROSCI.5086-09.2010

    Figure Lengend Snippet: ( A ) Fura-2 imaging of Ca ++ responses to 20mM K + in primary cultures of OPCs. The time of addition of high K + containing external solution is indicated by the horizontal bar. Note that each trace corresponds to a single cell. ( B ) K + -induced Ca ++ uptake was increased in 5μM BayK and abolished in 10μM Cd ++ , 25μM verapamil, 25μM nifedipine and in the absence of external Ca ++ (-Ca ++ ). The graph shows the average amplitude (peak) calculated from the responding cells, expressed as percentage of change of the emission intensities. Each agonist was applied by a fast and local perfusion system. Values are expressed as mean ± SEM of at least four independent experiments ( n > 500 cells for each condition). **p

    Article Snippet: The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments, Novato, CA).

    Techniques: Imaging

    ( A ) Fura-2 imaging of Ca ++ response to 20mM K + in control OPCs (Basal). Effect of genistein (10μM) ( B ), and the tyrosine phosphatase inhibitor, orthovanadate (5μM) ( C ), on Ca ++ influx induced by high K + in OPCs. The time of addition of high K + containing external solution and the TK modulators is indicated by the horizontal bars. ( D ) The graph show the average maximal peak values and plateau values during minutes 5-7, calculated from the responding cells, expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Multiple kinase pathways regulate voltage-dependent Ca++ influx and migration in oligodendrocyte precursor cells

    doi: 10.1523/JNEUROSCI.5086-09.2010

    Figure Lengend Snippet: ( A ) Fura-2 imaging of Ca ++ response to 20mM K + in control OPCs (Basal). Effect of genistein (10μM) ( B ), and the tyrosine phosphatase inhibitor, orthovanadate (5μM) ( C ), on Ca ++ influx induced by high K + in OPCs. The time of addition of high K + containing external solution and the TK modulators is indicated by the horizontal bars. ( D ) The graph show the average maximal peak values and plateau values during minutes 5-7, calculated from the responding cells, expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments ( n > 200 cells for each condition). *p

    Article Snippet: The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments, Novato, CA).

    Techniques: Imaging

    Emission spectra of RES and Fura-2. Separate measurements (A) of 340 nm (Ca 2+ -bound, blue) and 380 nm (Ca 2+ -free, orange) signal intensity in MDA cells with 5 µM TG and 100 µM RES treatments at 1 min and 6 min, respectively, as indicated by the arrows. This data (A) is representative of all experiments conducted at these concentrations. Emission spectra (B) in 1 mM Ca 2+ solution when excited at 340 nm of 100 µM RES (blue), and 5 µM Fura-2 (yellow).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: Emission spectra of RES and Fura-2. Separate measurements (A) of 340 nm (Ca 2+ -bound, blue) and 380 nm (Ca 2+ -free, orange) signal intensity in MDA cells with 5 µM TG and 100 µM RES treatments at 1 min and 6 min, respectively, as indicated by the arrows. This data (A) is representative of all experiments conducted at these concentrations. Emission spectra (B) in 1 mM Ca 2+ solution when excited at 340 nm of 100 µM RES (blue), and 5 µM Fura-2 (yellow).

    Article Snippet: Fura-2, pentasodium salt (50032) was purchased from Biotium (Hayward, CA).

    Techniques: Multiple Displacement Amplification

    PMCA inhibition by RES is unique among polyphenols. Structures of resveratrol (A), quercetin (B), and epigallocatechin gallate (EGCG) (C). PDF cells (D) were imaged for 15 min using Fura-2. Arrows represent treatment with 5 µM TG, and either 100 µM RES (black), 100 µM quercetin (blue), 100 µM EGCG (red) or vehicle (green) at 1 min and 6 min, respectively. Quercetin, EGCG and vehicle all showed statistically significant differences in [Ca 2+ ] i changes from RES in PDF cells (* signifies a statistically significant difference from RES at p

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: PMCA inhibition by RES is unique among polyphenols. Structures of resveratrol (A), quercetin (B), and epigallocatechin gallate (EGCG) (C). PDF cells (D) were imaged for 15 min using Fura-2. Arrows represent treatment with 5 µM TG, and either 100 µM RES (black), 100 µM quercetin (blue), 100 µM EGCG (red) or vehicle (green) at 1 min and 6 min, respectively. Quercetin, EGCG and vehicle all showed statistically significant differences in [Ca 2+ ] i changes from RES in PDF cells (* signifies a statistically significant difference from RES at p

    Article Snippet: Fura-2, pentasodium salt (50032) was purchased from Biotium (Hayward, CA).

    Techniques: Inhibition

    The direct effects of RES on Fura-2 fluorescence. Solutions containing 5 µM Fura-2 pentasodium salt were titrated with RES at 0 µM (blue), 1 µM (orange), 10 µM (gray), 25 µM (yellow), 50 µM (red), 75 µM (green), and 100 µM (purple). The samples were excited at 340 nm and 380 nm and the emission of each sample was measured at 510 nm. Each measurement was performed in triplicate in PBS with 0 mM Ca 2+ or 1 mM Ca 2+ as outlined in the figure (*signifies a statistically significant difference from Fura-2 emission at 510 nm at p

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: The direct effects of RES on Fura-2 fluorescence. Solutions containing 5 µM Fura-2 pentasodium salt were titrated with RES at 0 µM (blue), 1 µM (orange), 10 µM (gray), 25 µM (yellow), 50 µM (red), 75 µM (green), and 100 µM (purple). The samples were excited at 340 nm and 380 nm and the emission of each sample was measured at 510 nm. Each measurement was performed in triplicate in PBS with 0 mM Ca 2+ or 1 mM Ca 2+ as outlined in the figure (*signifies a statistically significant difference from Fura-2 emission at 510 nm at p

    Article Snippet: Fura-2, pentasodium salt (50032) was purchased from Biotium (Hayward, CA).

    Techniques: Fluorescence

    PMCA activity is inhibited by RES. Cells were monitored for changes in [Ca 2+ ] i for 15 min using Fura-2. Graphs depict changes in [Ca 2+ ] i as measured by 340 nm/380 nm ratio signal intensity. PMCA inhibition with LaCl 3 (A) (arrows mark addition of 5 µM TG and 1 mM LaCl 3 at 1 min and 6 min, respectively). LaCl 3 induced an increase in [Ca 2+ ] i in PDF (blue) and MDA (black) cell types as compared to vehicle only treatment in the respective cell types (PDF cells represented by green, MDA cells represented by red), indicative of PMCA inhibition. To ensure there were no calcium independent effects, BAPTA and Fura-2 were co-loaded into cells (B) (arrows mark addition of 5 µM TG and 50 µM RES at 1 min and 6 min, respectively). PMCA inhibition by RES (C), (arrows mark addition of 5 µM TG and 100 µM RES at 1 min and 6 min, respectively) in both PDF (blue) and MDA (black) cells. RES induced a statistically significant rise in [Ca 2+ ] i as compared to a vehicle only treatment (* signifies a statistically significant difference from respective vehicle controls at p

    Journal: Biochemistry and Biophysics Reports

    Article Title: Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

    doi: 10.1016/j.bbrep.2016.06.019

    Figure Lengend Snippet: PMCA activity is inhibited by RES. Cells were monitored for changes in [Ca 2+ ] i for 15 min using Fura-2. Graphs depict changes in [Ca 2+ ] i as measured by 340 nm/380 nm ratio signal intensity. PMCA inhibition with LaCl 3 (A) (arrows mark addition of 5 µM TG and 1 mM LaCl 3 at 1 min and 6 min, respectively). LaCl 3 induced an increase in [Ca 2+ ] i in PDF (blue) and MDA (black) cell types as compared to vehicle only treatment in the respective cell types (PDF cells represented by green, MDA cells represented by red), indicative of PMCA inhibition. To ensure there were no calcium independent effects, BAPTA and Fura-2 were co-loaded into cells (B) (arrows mark addition of 5 µM TG and 50 µM RES at 1 min and 6 min, respectively). PMCA inhibition by RES (C), (arrows mark addition of 5 µM TG and 100 µM RES at 1 min and 6 min, respectively) in both PDF (blue) and MDA (black) cells. RES induced a statistically significant rise in [Ca 2+ ] i as compared to a vehicle only treatment (* signifies a statistically significant difference from respective vehicle controls at p

    Article Snippet: Fura-2, pentasodium salt (50032) was purchased from Biotium (Hayward, CA).

    Techniques: Activity Assay, Inhibition, Multiple Displacement Amplification

    J.SL1 cells lack expression of Shc and are defective in IL-2 production. ( A ) Cell lysates of Jurkat and J.SL1 cells were analyzed by Western blots with the antibodies shown on the side of each panel. ( B ) Northern blot for lck and shc mRNA. Total RNA from Jurkat and J.SL1 cells was analyzed by using the cDNA fragment for lck ( Left ) or shc ( Right ). ( C ) IL-2 production by Jurkat and J.SL1 cells. Cells were cultured 16 h with (TCR) or without (−) stimulation by anti-TCR ( Left ) or with PMA and ionomycin (P+I, Right ). The amount of IL-2 in the culture supernatant from each sample was determined by ELISA. A representative result of several experiments is shown. ( D ) Surface expression of CD69 and CD25 on Jurkat and J.SL1 cells. Jurkat and J.SL1 cells were treated as described for C , and expression of CD69 and CD25 were examined by using FITC-conjugated antibodies. The dark lines show fluorescence levels detected from activated cells, and gray lines show that from unstimulated cells. ( E ) Induction of intracellular Ca 2+ in Jurkat and J.SL1 cells. Jurkat ( Left ) and J.SL1 ( Right ) cells were loaded with Fura-2 acetoxymethyl ester, and the cells were stimulated with anti-TCR at the times shown by the arrow above each panel. The level of Ca 2+ was determined on the basis of the ratio between fluorescence from excitation at 340 nm to that from excitation at 380 nm.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression

    doi: 10.1073/pnas.082647499

    Figure Lengend Snippet: J.SL1 cells lack expression of Shc and are defective in IL-2 production. ( A ) Cell lysates of Jurkat and J.SL1 cells were analyzed by Western blots with the antibodies shown on the side of each panel. ( B ) Northern blot for lck and shc mRNA. Total RNA from Jurkat and J.SL1 cells was analyzed by using the cDNA fragment for lck ( Left ) or shc ( Right ). ( C ) IL-2 production by Jurkat and J.SL1 cells. Cells were cultured 16 h with (TCR) or without (−) stimulation by anti-TCR ( Left ) or with PMA and ionomycin (P+I, Right ). The amount of IL-2 in the culture supernatant from each sample was determined by ELISA. A representative result of several experiments is shown. ( D ) Surface expression of CD69 and CD25 on Jurkat and J.SL1 cells. Jurkat and J.SL1 cells were treated as described for C , and expression of CD69 and CD25 were examined by using FITC-conjugated antibodies. The dark lines show fluorescence levels detected from activated cells, and gray lines show that from unstimulated cells. ( E ) Induction of intracellular Ca 2+ in Jurkat and J.SL1 cells. Jurkat ( Left ) and J.SL1 ( Right ) cells were loaded with Fura-2 acetoxymethyl ester, and the cells were stimulated with anti-TCR at the times shown by the arrow above each panel. The level of Ca 2+ was determined on the basis of the ratio between fluorescence from excitation at 340 nm to that from excitation at 380 nm.

    Article Snippet: Cells were loaded with Fura-2 acetoxymethyl ester (Dojindo, Kumamoto, Japan), and the level of Ca2+ was determined on the basis of the ratio between fluorescence from excitation at 340 nm to that from excitation at 380 nm by using a spectrofluorometer RF1500 (Shimadzu).

    Techniques: Expressing, Western Blot, Northern Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

    ATP (0.1 mM) produced a prompt increase in the fura 2 fluorescence ratio in MDCK monolayers perfused with solution containing 2 mM Ca 2+ ( top, left ). This was due in part to increased Ca 2+ influx because ATP stimulated rapid quenching of fura 2 by extracellular Mn 2+ (2 mM) in the absence of extracellular Ca 2+ ( top, right ). ATP treatment in Ca 2+ -free solution also increased the fluorescence ratio, suggesting mobilization of intracellular Ca 2+ stores ( bottom ). This was followed by a second fluorescence peak when extracellular calcium was restored, consistent with Ca 2+ influx. A single curve is shown that represents the mean ratio derived from the number of cells indicated on the graphs.

    Journal: American Journal of Physiology. Renal Physiology

    Article Title: Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

    doi: 10.1152/ajprenal.00108.2008

    Figure Lengend Snippet: ATP (0.1 mM) produced a prompt increase in the fura 2 fluorescence ratio in MDCK monolayers perfused with solution containing 2 mM Ca 2+ ( top, left ). This was due in part to increased Ca 2+ influx because ATP stimulated rapid quenching of fura 2 by extracellular Mn 2+ (2 mM) in the absence of extracellular Ca 2+ ( top, right ). ATP treatment in Ca 2+ -free solution also increased the fluorescence ratio, suggesting mobilization of intracellular Ca 2+ stores ( bottom ). This was followed by a second fluorescence peak when extracellular calcium was restored, consistent with Ca 2+ influx. A single curve is shown that represents the mean ratio derived from the number of cells indicated on the graphs.

    Article Snippet: Fura 2 was excited by alternating 340 and 380 nm light, and fluorescence emitted at 510 nm was collected by a CCD camera attached to a computer for data acquisition by the InCa ratiometric fluorescence program.

    Techniques: Produced, Fluorescence, Derivative Assay

    Top : time course of changes in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP in MDCK control cells ( left ) and in cells preloaded for 30 min with 25 μM BAPTA-AM ( right ). Extracellular solution contained 2 mM Ca 2+ . Bottom : ATP produced no significant change in the fluorescence ratio in BAPTA-loaded cells (means ± SE of 30 cells). *Significantly different ( P

    Journal: American Journal of Physiology. Renal Physiology

    Article Title: Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

    doi: 10.1152/ajprenal.00108.2008

    Figure Lengend Snippet: Top : time course of changes in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP in MDCK control cells ( left ) and in cells preloaded for 30 min with 25 μM BAPTA-AM ( right ). Extracellular solution contained 2 mM Ca 2+ . Bottom : ATP produced no significant change in the fluorescence ratio in BAPTA-loaded cells (means ± SE of 30 cells). *Significantly different ( P

    Article Snippet: Fura 2 was excited by alternating 340 and 380 nm light, and fluorescence emitted at 510 nm was collected by a CCD camera attached to a computer for data acquisition by the InCa ratiometric fluorescence program.

    Techniques: Fluorescence, Produced

    SU-DHL-4 cells are protected from BIRD-2-triggered apoptosis by genetically manipulating the IP 3 signaling pathway. a Examples of flow cytometry analysis showing the percentage of GFP-positive transfected SU-DHL-4 cells, visible as a shift in the BL-1 (515–545 nm) channel, while the values in the BL-3 (665–715 nm) channel remained unaffected. b Representative flow cytometry analysis of BIRD-2-induced apoptosis in SU-DHL-4 cells transfected with the IP 3 sponge or a control vector compared to mock-transfected cells. Apoptosis was detected via Annexin V-APC-positive staining (RL1 + = red laser, see Method section) of the cells. c Quantification of the apoptotic fraction (%) after treatment with 10 µM BIRD-2 (red histogram in panel b ) or vehicle (black histogram panel b ) in mock-transfected SU-DHL-4 cells or cells transfected with the IP 3 sponge or a control vector. Apoptotic cells were identified as the Annexin V-APC-positive fraction (RL1 + ). Data are represented as mean ± SEM of 3 independent experiments. d Single-cell cytosolic Ca 2+ measurements performed in SU-DHL-4 cells utilizing Fura-2 AM. Cells were transfected with an IP 3 sponge vector or with an empty vector as negative control condition (pcDNA3.1). The addition of 10 µM BIRD-2 is indicated by the arrow. Data are represented as the average ± SEM of 3 independent experiments ( n > 100 cells/condition)

    Journal: Cell Death and Differentiation

    Article Title: Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2

    doi: 10.1038/s41418-018-0142-3

    Figure Lengend Snippet: SU-DHL-4 cells are protected from BIRD-2-triggered apoptosis by genetically manipulating the IP 3 signaling pathway. a Examples of flow cytometry analysis showing the percentage of GFP-positive transfected SU-DHL-4 cells, visible as a shift in the BL-1 (515–545 nm) channel, while the values in the BL-3 (665–715 nm) channel remained unaffected. b Representative flow cytometry analysis of BIRD-2-induced apoptosis in SU-DHL-4 cells transfected with the IP 3 sponge or a control vector compared to mock-transfected cells. Apoptosis was detected via Annexin V-APC-positive staining (RL1 + = red laser, see Method section) of the cells. c Quantification of the apoptotic fraction (%) after treatment with 10 µM BIRD-2 (red histogram in panel b ) or vehicle (black histogram panel b ) in mock-transfected SU-DHL-4 cells or cells transfected with the IP 3 sponge or a control vector. Apoptotic cells were identified as the Annexin V-APC-positive fraction (RL1 + ). Data are represented as mean ± SEM of 3 independent experiments. d Single-cell cytosolic Ca 2+ measurements performed in SU-DHL-4 cells utilizing Fura-2 AM. Cells were transfected with an IP 3 sponge vector or with an empty vector as negative control condition (pcDNA3.1). The addition of 10 µM BIRD-2 is indicated by the arrow. Data are represented as the average ± SEM of 3 independent experiments ( n > 100 cells/condition)

    Article Snippet: Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127).

    Techniques: Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Staining, Negative Control

    SU-DHL-4 cells display constitutive IP 3 /Ca 2+ signaling. a The basal Ca 2+ level was used as a read-out for measuring the level of constitutive IP 3 ]. PLC activity was suppressed using U73122, whereas its inactive enantiomer U73343 did not affect PLC activity. b A typical fluorescent recording of the basal [Ca 2+ ] cyt in SU-DHL-4 cells pre-treated with vehicle (black line), 1 μM U73122 (red line) or 1 µM U73343 (gray line) using the ratiometric Ca 2+ indicator Fura-2 AM. The cells were present in Krebs medium supplemented with 1.5 mM CaCl 2 . The ratio of emitted fluorescence of Fura-2 (F 340 /F 380 ) was monitored and Ca 2+ values were calibrated by adding digitonin (50 µM) and a 20-fold excess of EGTA (33 mM) to determine R max and R min respectively (see Method section). Basal [Ca 2+ ] (nM) are reported in c as the mean ± SEM ( N = 5). The exact values of each independent experiment are represented in different colors. Statistically significant differences were determined using an analysis of variance (ANOVA, ** P

    Journal: Cell Death and Differentiation

    Article Title: Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2

    doi: 10.1038/s41418-018-0142-3

    Figure Lengend Snippet: SU-DHL-4 cells display constitutive IP 3 /Ca 2+ signaling. a The basal Ca 2+ level was used as a read-out for measuring the level of constitutive IP 3 ]. PLC activity was suppressed using U73122, whereas its inactive enantiomer U73343 did not affect PLC activity. b A typical fluorescent recording of the basal [Ca 2+ ] cyt in SU-DHL-4 cells pre-treated with vehicle (black line), 1 μM U73122 (red line) or 1 µM U73343 (gray line) using the ratiometric Ca 2+ indicator Fura-2 AM. The cells were present in Krebs medium supplemented with 1.5 mM CaCl 2 . The ratio of emitted fluorescence of Fura-2 (F 340 /F 380 ) was monitored and Ca 2+ values were calibrated by adding digitonin (50 µM) and a 20-fold excess of EGTA (33 mM) to determine R max and R min respectively (see Method section). Basal [Ca 2+ ] (nM) are reported in c as the mean ± SEM ( N = 5). The exact values of each independent experiment are represented in different colors. Statistically significant differences were determined using an analysis of variance (ANOVA, ** P

    Article Snippet: Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127).

    Techniques: Planar Chromatography, Activity Assay, Fluorescence

    U73122 reduces the BIRD-2-induced cytosolic Ca 2+ rise in SU-DHL-4 cells. a Single-cell analysis of the BIRD-2-induced Ca 2+ response in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Representative pseudo-color images before (2 s) and after (500 s) BIRD-2 treatment are shown. Vehicle and TAT-Ctrl were used as negative control conditions. The pseudo-color scale bar indicates increasing ratio fluorescence. b Single-cell cytosolic Ca 2+ signals (gray lines) and their respective mean (black line) upon addition of vehicle, TAT-ctrl peptide or 10 µM BIRD-2 to SU-DHL-4 cells without or with pre-treatment of 1 μM U73122/U73343. c Cell-population analysis of the cytosolic Ca 2+ response induced by 10 µM BIRD-2 in SU-DHL-4 cells pre-treated without (black line) or with 1 µM U73122 (green line) or 1 µM U73343 (gray line). The curves are representative of 4 independent experiments. Data were quantified by calculating the peak amplitude ( d ). In d , data are represented as mean ± SEM ( N = 4). Statistically significant differences were determined using an analysis of variance (ANOVA, * P

    Journal: Cell Death and Differentiation

    Article Title: Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2

    doi: 10.1038/s41418-018-0142-3

    Figure Lengend Snippet: U73122 reduces the BIRD-2-induced cytosolic Ca 2+ rise in SU-DHL-4 cells. a Single-cell analysis of the BIRD-2-induced Ca 2+ response in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Representative pseudo-color images before (2 s) and after (500 s) BIRD-2 treatment are shown. Vehicle and TAT-Ctrl were used as negative control conditions. The pseudo-color scale bar indicates increasing ratio fluorescence. b Single-cell cytosolic Ca 2+ signals (gray lines) and their respective mean (black line) upon addition of vehicle, TAT-ctrl peptide or 10 µM BIRD-2 to SU-DHL-4 cells without or with pre-treatment of 1 μM U73122/U73343. c Cell-population analysis of the cytosolic Ca 2+ response induced by 10 µM BIRD-2 in SU-DHL-4 cells pre-treated without (black line) or with 1 µM U73122 (green line) or 1 µM U73343 (gray line). The curves are representative of 4 independent experiments. Data were quantified by calculating the peak amplitude ( d ). In d , data are represented as mean ± SEM ( N = 4). Statistically significant differences were determined using an analysis of variance (ANOVA, * P

    Article Snippet: Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127).

    Techniques: Single-cell Analysis, Negative Control, Fluorescence

    U73122 protects against BIRD-2-triggered apoptosis in SU-DHL-4. a Cell-population analysis of the cytosolic Ca 2+ response, measured with Fura-2 AM, in SU-DHL-4 cells pre-treated for 30 min with U73122 (1 and 2.5 µM), U73343 (2.5 µM) or vehicle (DMSO). Addition of 3 mM EGTA and 12 µg/ml anti-IgG/M antibody is indicated by the first and second arrow, respectively. The curves are representative of 3 independent experiments. The cytosolic Ca 2+ response after anti-IgG/M addition was quantified by measuring the area under the curve (AUC), which is shown in b . c Quantitative analysis of 3 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells. Cells were treated with varying concentrations of U73122 or 2.5 µM U73343. Apoptotic cell death was measured 30 min, 2 h and 24 h after treatment. On the y-axis the percentage of living cells is plotted. Data are shown as the average ± SEM ( N = 3). d Representative dot plots from flow cytometry analysis detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells treated for 2 h with vehicle or 10 µM BIRD-2. Cells were pre-treated for 30 min with U73122 or U73343. e, f Quantitative analysis of 4 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells. Apoptotic cell death was measured as the percentage of Annexin V-FITC-positive cells. Cells were pre-treated with U73122 or U73343 for 30 min. Cell death was measured 2 h ( e ) and 24 h ( f ) after BIRD-2 treatment. On the y-axis, the ∆ apoptotic fraction is plotted, which is the difference in apoptosis between the BIRD-2-treated and the vehicle-treated fraction, and between the BIRD-2 + U73122-treated and the U73122-treated fraction, and finally between the BIRD-2 + U73343-treated and the U73343-treated fraction. Data are shown as the average ± SEM ( N = 5). g Quantitative analysis of 4 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells treated with 1 or 2.5 µM U73122, 2.5 µM U73343, 5 µM BIRD-2 (blue), 3 µM venetoclax (green) or a combination of U73122/U73343 with BIRD-2/venetoclax. For the conditions without Bcl-2 inhibitor (indicated with a ‘-’), the green bars indicate the use of the vehicle control for venetoclax, while the blue bars indicate the use of vehicle control for BIRD-2 treatment. A ‘+’ indicates that the Bcl-2 inhibitor (BIRD-2/venetoclax) was added in this condition. Cell death was measured 24 h after treatment. On the y-axis the percentage of living cells, which corresponds to the Annexin V-FITC- and 7-AAD-negative fraction, is shown. Data are expressed as the average ± SEM ( N = 4). h CI derived from SU-DHL-4 cells treated with U73122/U73343 in combination with BIRD-2/venetoclax. The CI was calculated (see Method section) from the data shown in g . Statistically significant differences were determined using an analysis of variance (ANOVA, * P

    Journal: Cell Death and Differentiation

    Article Title: Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2

    doi: 10.1038/s41418-018-0142-3

    Figure Lengend Snippet: U73122 protects against BIRD-2-triggered apoptosis in SU-DHL-4. a Cell-population analysis of the cytosolic Ca 2+ response, measured with Fura-2 AM, in SU-DHL-4 cells pre-treated for 30 min with U73122 (1 and 2.5 µM), U73343 (2.5 µM) or vehicle (DMSO). Addition of 3 mM EGTA and 12 µg/ml anti-IgG/M antibody is indicated by the first and second arrow, respectively. The curves are representative of 3 independent experiments. The cytosolic Ca 2+ response after anti-IgG/M addition was quantified by measuring the area under the curve (AUC), which is shown in b . c Quantitative analysis of 3 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells. Cells were treated with varying concentrations of U73122 or 2.5 µM U73343. Apoptotic cell death was measured 30 min, 2 h and 24 h after treatment. On the y-axis the percentage of living cells is plotted. Data are shown as the average ± SEM ( N = 3). d Representative dot plots from flow cytometry analysis detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells treated for 2 h with vehicle or 10 µM BIRD-2. Cells were pre-treated for 30 min with U73122 or U73343. e, f Quantitative analysis of 4 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells. Apoptotic cell death was measured as the percentage of Annexin V-FITC-positive cells. Cells were pre-treated with U73122 or U73343 for 30 min. Cell death was measured 2 h ( e ) and 24 h ( f ) after BIRD-2 treatment. On the y-axis, the ∆ apoptotic fraction is plotted, which is the difference in apoptosis between the BIRD-2-treated and the vehicle-treated fraction, and between the BIRD-2 + U73122-treated and the U73122-treated fraction, and finally between the BIRD-2 + U73343-treated and the U73343-treated fraction. Data are shown as the average ± SEM ( N = 5). g Quantitative analysis of 4 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-4 cells treated with 1 or 2.5 µM U73122, 2.5 µM U73343, 5 µM BIRD-2 (blue), 3 µM venetoclax (green) or a combination of U73122/U73343 with BIRD-2/venetoclax. For the conditions without Bcl-2 inhibitor (indicated with a ‘-’), the green bars indicate the use of the vehicle control for venetoclax, while the blue bars indicate the use of vehicle control for BIRD-2 treatment. A ‘+’ indicates that the Bcl-2 inhibitor (BIRD-2/venetoclax) was added in this condition. Cell death was measured 24 h after treatment. On the y-axis the percentage of living cells, which corresponds to the Annexin V-FITC- and 7-AAD-negative fraction, is shown. Data are expressed as the average ± SEM ( N = 4). h CI derived from SU-DHL-4 cells treated with U73122/U73343 in combination with BIRD-2/venetoclax. The CI was calculated (see Method section) from the data shown in g . Statistically significant differences were determined using an analysis of variance (ANOVA, * P

    Article Snippet: Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127).

    Techniques: Staining, Flow Cytometry, Cytometry, Derivative Assay

    rgs3 impacts segmentation stage Ca 2+ dynamics. Zebrafish embryos injected with Fura-2 were oriented in a dorsal posterior view. Representative ratio images, pseudocolored with low Ca 2+ represented by blue, and high Ca 2+ represented by yellow/red (A–C,F–N). During somitogenesis, Ca 2+ transients are identified as a local short-lived increase in intracellular Ca 2+ levels. A region of interest (ROI) is noted by a dashed circle highlighting a representative Ca 2+ transient (A–C). In the ROI from time 0s to time 15s, an increase in Ca 2+ levels is observed (B) that subsides by time 30s (C). The number of transients as a function of developmental age (D). Table depicting the average number of Ca 2+ transients per hour from 6 to 12 somite stage for each treatment (E). Representative ratio images of 5 somite stage (F), 7 somite (G) and 10 somite stage (H) wt embryos taken from Video S1 . Representative ratio images of 5 somite (I), 7 somite (J) and 10 somite stage (K) rgs3 MOsa injected embryo taken from Video S2 . Representative ratio images of 5 somite (L), 7 somite stage (M) and 10 somite stage (N) wnt5b MO injected embryo taken from Video S4 . Red arrowheads indicate large Ca 2+ transients in rgs3 morphant embryos (I–K) that are not observed in wt (F–H) or wnt5b morphant embryos (L–N).

    Journal: PLoS Genetics

    Article Title: Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

    doi: 10.1371/journal.pgen.1001020

    Figure Lengend Snippet: rgs3 impacts segmentation stage Ca 2+ dynamics. Zebrafish embryos injected with Fura-2 were oriented in a dorsal posterior view. Representative ratio images, pseudocolored with low Ca 2+ represented by blue, and high Ca 2+ represented by yellow/red (A–C,F–N). During somitogenesis, Ca 2+ transients are identified as a local short-lived increase in intracellular Ca 2+ levels. A region of interest (ROI) is noted by a dashed circle highlighting a representative Ca 2+ transient (A–C). In the ROI from time 0s to time 15s, an increase in Ca 2+ levels is observed (B) that subsides by time 30s (C). The number of transients as a function of developmental age (D). Table depicting the average number of Ca 2+ transients per hour from 6 to 12 somite stage for each treatment (E). Representative ratio images of 5 somite stage (F), 7 somite (G) and 10 somite stage (H) wt embryos taken from Video S1 . Representative ratio images of 5 somite (I), 7 somite (J) and 10 somite stage (K) rgs3 MOsa injected embryo taken from Video S2 . Representative ratio images of 5 somite (L), 7 somite stage (M) and 10 somite stage (N) wnt5b MO injected embryo taken from Video S4 . Red arrowheads indicate large Ca 2+ transients in rgs3 morphant embryos (I–K) that are not observed in wt (F–H) or wnt5b morphant embryos (L–N).

    Article Snippet: Intracellular calcium (Ca2+ ) imaging The ratiometric Ca2+ -sensing dye Fura-2 or Bis-Fura-2 (Molecular Probes) was injected into 1-cell zebrafish embryos.

    Techniques: Injection

    rgs3 inhibits wnt5b- induced Ca 2+ dynamics. Schematic representation illustrating that Wnt5b overexpression results in intracellular calcium release in a G protein dependent manner (A, left side) and the predicted negative effect overexpression of Rgs3 will have on the Wnt/calcium pathway (A, right side). Representative Ca 2+ release profiles (composite image) of wnt5- overexpressing (B,D,E) and wt (C) blastula stage zebrafish embryos. (B–E) are composites of fura-2 ratiometric imaging time course showing total calcium release activity as peaks and colors mapped topographically. Ca 2+ release profile of an embryo uniformly expressing wnt5b (B). Wt Ca 2+ release profile (C). wnt5b expressing embryo with localized TxR/ rgs3 (D) or Txr/ rgs3 N109A (E). Corresponding fluorescent images illustrate the location of TxR/ rgs3 (F) and TxR/ rgs3 N109A (G).

    Journal: PLoS Genetics

    Article Title: Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

    doi: 10.1371/journal.pgen.1001020

    Figure Lengend Snippet: rgs3 inhibits wnt5b- induced Ca 2+ dynamics. Schematic representation illustrating that Wnt5b overexpression results in intracellular calcium release in a G protein dependent manner (A, left side) and the predicted negative effect overexpression of Rgs3 will have on the Wnt/calcium pathway (A, right side). Representative Ca 2+ release profiles (composite image) of wnt5- overexpressing (B,D,E) and wt (C) blastula stage zebrafish embryos. (B–E) are composites of fura-2 ratiometric imaging time course showing total calcium release activity as peaks and colors mapped topographically. Ca 2+ release profile of an embryo uniformly expressing wnt5b (B). Wt Ca 2+ release profile (C). wnt5b expressing embryo with localized TxR/ rgs3 (D) or Txr/ rgs3 N109A (E). Corresponding fluorescent images illustrate the location of TxR/ rgs3 (F) and TxR/ rgs3 N109A (G).

    Article Snippet: Intracellular calcium (Ca2+ ) imaging The ratiometric Ca2+ -sensing dye Fura-2 or Bis-Fura-2 (Molecular Probes) was injected into 1-cell zebrafish embryos.

    Techniques: Over Expression, Imaging, Activity Assay, Expressing